Methods and compositions to enhance activity of cry endotoxins

ABSTRACT

Methods and compositions for enhancing the resistance of plants to plant pests are provided. Chimeric pesticidal polypeptides and nucleic acid molecules encoding the chimeric pesticidal polypeptides are provided. The chimeric pesticidal polypeptides comprising a solubility-enhancing polypeptide operably linked to a polypeptide comprising pesticidal activity. The nucleic acid molecules can be used in expression cassettes for making transformed plants with enhanced resistance to plant pests. Further provided are transformed plants, plant tissues, plant cells, other host cells, and seeds as well as pesticidal compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit U.S. Provisional Application No.61/713,844, filed Oct. 15, 2012, which is incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

An official copy of the Sequence Listing submitted electronically as anASCII formatted Sequence Listing with a file named“3113PCT_SequenceListing.txt,” created on Sep. 17, 2013, having a sizeof 158 kb and filed concurrently with the Specification is part of theSpecification and is incorporated herein by reference as if set forth inits entirety.

FIELD OF THE INVENTION

The invention relates generally to plant molecular biology and plantpest control, and more particularly to compositions and methods forenhancing the activity of pesticidal polypeptides from Bacillus spp. andfor protecting a plant from a plant pest, particularly an insect pest.

BACKGROUND OF THE INVENTION

Pests, such as insect pests, are a major factor in the loss of theworld's agricultural crops. For example, corn rootworm and boll weevildamage can be economically devastating to agricultural producers. Insectpest-related agricultural crop loss from corn rootworm alone has reachedone billion dollars a year.

Traditionally, the primary methods for controlling insect pests, such ascorn rootworm, are crop rotation and application of broad-spectrum,synthetic, chemical pesticides. However, consumers and governmentregulators alike are becoming increasingly concerned with environmentalhazards associated with producing and using chemical pesticides. Becauseof such concerns, regulators have banned or limited the use of some ofthe more hazardous chemical pesticides. Thus, there is substantialinterest in developing alternatives to chemical pesticides that presenta lower risk of pollution and environmental hazards and that provide agreater target specificity than is characteristic of chemicalpesticides.

Certain species in the genus Bacillus have polypeptides that possesspesticidal activity against a broad range of insect pests includingthose in the orders Lepidoptera, Diptera, Coleoptera, Hemiptera andothers. For example, Bacillus thuringiensis and Bacillus popilliae areamong the most successful species discovered to date having pesticidalactivity. Such pesticidal activity also has been attributed to strainsof Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus andBacillus cereus. See, Biotechnology Handbook 2: Bacillus (Harwood ed.,Plenum Press 1989); and Int'l Patent Application Publication No. WO96/10083.

Pesticidal polypeptides from Bacillus spp. include the crystal (Cry)endotoxins, cytolytic (Cyt) endotoxins, vegetative proteins (VIPs) andthe like. See, e.g., Bravo et al. (2007) Toxicon 49:423-435. The Cryendotoxins (also called δ-endotoxins) have been isolated from variousstrains of B. thuringiensis. A common characteristic of the Cryendotoxins is their expression during the stationary phase of growth, asthey generally accumulate in a mother cell compartment to form a crystalinclusion that can account for 23-30% of the dry weight of sporulatedcells. The Cry endotoxins initially are produced in an inactive protoxinform, which are proteolytically converted into an active endotoxinthrough the action of proteases in an insect's gut. Once active, theendotoxins bind to the gut epithelium and form cation-selective channelsthat cause cell lysis and subsequent death. See, Carroll et al. (1997)J. Invertebr. Pathol. 70:41-49; Oppert (1999) Arch. Insect Biochem.Phys. 42:1-12; and Rukmini et al. (2000) Biochimie 82:109-116.

Although Cry endotoxins often are highly effective against insect pests,some insect pests are not affected by them or show low susceptibility.Likewise, some insect pests have developed resistance to the Cryendotoxins, which threatens their effectiveness. Methods to addressthese problems include enhancing or expanding Cry endotoxin activity bysite-directed mutagenesis, by introducing cleavage sites in specificregions of the endotoxin or by deleting small fragments from theamino-terminus of the endotoxin. See, e.g., Abdullah & Dean (2004) Appl.Environ. Microbiol. 70:3769-3771; Pardo-López et al. (2009) Peptides30:589-595; Rajamohan et al. (1996) Proc. Natl. Acad. Sci. USA93:14338-14343; and Wu et al. (2000) FEBS Lett. 473 227-232. Thesemethods, however, can be time consuming and not certain to produce adesired result.

For the foregoing reasons, there is a need for compositions and methodsto enhance the pesticidal activity of Cry endotoxins.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods are provided for enhancing pesticidal activityof Cry endotoxins and for protecting a plant from plant pest such asinsect pests. The compositions comprise chimeric pesticidalpolypeptide-encoding nucleic acid molecules, variants and fragmentsthereof, as well as chimeric pesticidal polypeptides, active variantsand fragments thereof. The chimeric pesticidal polypeptides of theinvention comprise a solubility-enhancing polypeptide fused to a Cryendotoxin or biologically active fragment thereof. Also provided areexpression cassettes or polynucleotide constructs comprising anucleotide sequence encoding a chimeric pesticidal polypeptide of theinvention, as well as bacteria, plants, plant organs, plants tissues,plant parts, plant cells and seeds comprising the expression cassette orpolynucleotide nucleotide construct encoding the chimeric pesticidalpolypeptide. Further provided are pesticidal compositions comprising atleast one chimeric pesticidal polypeptide of the invention.

Methods are provided for enhancing the pesticidal activity of Cryendotoxins. The methods involve making a chimeric pesticidal polypeptidecomprising the amino acid sequence of a solubility-enhancing polypeptideoperably linked to an amino acid sequence of a Cry endotoxin orbiologically active fragment thereof. Such chimeric pesticidalpolypeptide can be produced by fusing amino acid sequence of thesolubility-enhancing polypeptide to the amino acid sequence of a Cryendotoxin or biologically active fragment thereof. Alternatively, themethods involve making a polynucleotide construct comprising anucleotide sequence encoding the chimeric pesticidal polypeptide andtransforming an organism or non-human host cell of interest with thepolynucleotide construct for expression of the chimeric pesticidalpolypeptide. The nucleotide sequence encoding the chimeric pesticidalpolypeptide can be produced by, for example, operably linking anucleotide sequence encoding a solubility-enhancing polypeptide to anucleotide sequence encoding a Cry endotoxin or biologically activefragment thereof. Typically, the polynucleotide construct furthercomprises a promoter that drives expression in the organism or hostcell, wherein the promoter is operably linked to the nucleotide sequenceencoding the chimeric pesticidal polypeptide.

Thus, methods are provided for producing a polynucleotide constructcomprising a nucleotide sequence encoding a chimeric pesticidalpolypeptide, which comprises an amino acid sequence ofsolubility-enhancing polypeptide fused to an amino acid sequence of aCry endotoxin or biologically active fragment thereof. The methodsinvolve operably linking a nucleotide sequence encodingsolubility-enhancing polypeptide to a nucleotide sequence encoding a Cryendotoxin or biologically active fragment thereof. The polynucleotideconstruct may additional comprise an operably linked promoter forexpression of the chimeric pesticidal polypeptide in a non-human hostcell of interest, particularly a plant cell. Such a polynucleotideconstruct finds use, for example, in methods for expressing the chimericpesticidal polypeptide in a plant transformed therewith.

The present invention further provides methods for making plants withenhanced resistance to at least one pest. The methods involvedtransforming a plant or at least one plant cell with a polynucleotideconstruct comprising a nucleotide sequence encoding a chimericpesticidal polypeptide of the invention. Typically, the nucleotidesequence encoding a chimeric pesticidal polypeptide will be operablylinked to a promoter that drives expression in a plant cell. The methodscan further involve regenerating the plant or the at least one plantcell into a transformed plant, wherein the regenerated plant expressesthe chimeric pesticidal polypeptide. Such a transformed plant comprisesenhanced resistance to at least one plant pest, particularly an insectpest, when compared to the resistance of a control plant.

The methods also involve applying a composition such as a pesticidalcomposition comprising the chimeric pesticidal polypeptide or activevariant or fragment thereof, to the environment of an insect pest,particularly on or in the vicinity of a plant by, for example, spraying,dusting, broadcasting or seed coating to protect the plant from theinsect pest.

Further provided are transformed plants, plant cells and other hostcells, and seeds comprising a nucleotide sequence encoding the chimericpesticidal polypeptide of the invention.

The following embodiments are encompassed by the present invention:

1. A method of enhancing pesticidal activity of a Cry endotoxin, themethod comprising operably linking a first amino acid sequence of asolubility-enhancing polypeptide to a second amino acid sequence of aCry endotoxin, whereby a chimeric pesticidal polypeptide is produced,the chimeric pesticidal polypeptide comprising the first amino acidsequence operably linked to second amino acid sequence.

2. The method of embodiment 1, wherein the solubility-enhancingpolypeptide is selected from the group consisting of a maltose-bindingprotein (MBP), a thioredoxin, a transcription elongation factor NusA, aglutathione-S-transferase (GST), a mistic, a small ubiquitin-relatedmodifier (SUMO), a protein disulfide isomerase DsbC, and athiol:disulfide interchange protein DsbD.

3. The method of embodiment 1 or 2, wherein the solubility-enhancingpolypeptide is a MBP.

4. The method of embodiment 3, wherein the MBP is selected from thegroup consisting of MBPs having an accession number set forth in Table1.

5. The method of embodiment 1 or 2, wherein the solubility-enhancingprotein is NusA.

6. The method of embodiment 1 or 2, wherein the solubility-enhancingprotein is thioredoxin.

7. The method of embodiment 1, wherein the solubility-enhancingpolypeptide comprises an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOS: 4, 6,34, 35, and 36.

8. The method of any one of embodiments 1-7, wherein the Cry endotoxinis selected from the group consisting of Cry endotoxins set forth inTable 2.

9. The method of any one of embodiments 1-8, wherein the Cry endotoxincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS: 8, 10, 12, and 14.

10. The method of embodiment 1, wherein the chimeric pesticidalpolypeptide comprises an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOS: 2, 16,18, 21, 23, 25, 27, and 33.

11. The method of embodiment 1, wherein the chimeric pesticidalpolypeptide is encoded by a nucleotide sequence comprising thenucleotide sequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26,and 32.

12. The method of any one of embodiments 1-11, wherein the chimericpesticidal polypeptide comprises a linker amino sequence between thefirst amino acid sequence and the second amino acid sequence.

13. The method of embodiment 1, wherein the linker amino acid sequenceis selected from the group consisting of the amino acid sequences setforth in SEQ ID NOS: 28, 29, 30, and 31.

14. The method of any one of embodiments 1-13, further comprisingoperably linking the linker amino acid sequence between the first aminoacid sequence and the amino acid sequence, whereby the chimericpesticidal polypeptide comprises in linear order the first amino acidsequence, the linker amino acid sequence, and the second amino acidsequence.

15. The method of any one of embodiments 1-15, wherein the chimericpesticidal polypeptide comprises increased pesticidal activity againstat least one pest, when compared to the pesticidal activity of the Cryendotoxin against the at least one pest.

16. The method of embodiment 15, wherein the at least one pest is aninsect pest.

17. The method of embodiment 16, wherein the insect pest is an insectpest from the order Coleoptera or Lepidoptera.

18. The method of embodiment 16 or 17, wherein the insect pest is fromthe genus Diabrotica.

19. The method of embodiment 16, wherein the insect pest is western cornrootworm.

20. The method of embodiment 16, wherein the insect pest is blackcutworm.

21. A chimeric pesticidal polypeptide comprising a first amino acidsequence of a solubility-enhancing polypeptide operably linked to asecond amino acid sequence of a Cry endotoxin.

22. The chimeric pesticidal polypeptide of embodiment 21, wherein theoperably linked first and second amino sequences comprise an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence set forth in SEQ ID NO: 2, 16, 18,        21, 23, 25, 27 or 33;    -   (b) an amino acid sequence encoded by the nucleotide sequence        set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32; and    -   (c) an amino acid sequence comprising at least 80% amino acid        sequence identity to the amino acid sequence set forth in SEQ ID        NO: 2, 16, 18, 21, 23, 25, 27 or 33, wherein the nucleotide        sequence encodes a polypeptide having pesticidal activity.

23. The chimeric pesticidal polypeptide of embodiment 21 or 22, whereinthe solubility-enhancing polypeptide is a MBP.

24. The chimeric pesticidal polypeptide of embodiment 23, wherein theMBP is selected from the group consisting of MBPs having an accessionnumber set forth in Table 1.

25. The chimeric pesticidal polypeptide of embodiment 21 or 22, whereinthe solubility-enhancing protein is a NusA.

26. The chimeric pesticidal polypeptide of embodiment 21 or 22, whereinthe solubility-enhancing protein is a thioredoxin.

27. The chimeric pesticidal polypeptide of embodiment 21, wherein thefirst amino acid sequence comprises an amino acid sequence selected fromthe group consisting of the amino acid sequences set forth in SEQ IDNOS: 4, 6, 34, 35, and 36.

28. The chimeric pesticidal polypeptide of any one of embodiments 21-27,wherein the Cry endotoxin is selected from the group consisting of Cryendotoxins set forth in Table 2.

29. The chimeric pesticidal polypeptide of embodiment 21, wherein theCry endotoxin comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 8, 10, 12, and 14.

30. The chimeric pesticidal polypeptide of any one of embodiments 21-29,wherein the chimeric pesticidal polypeptide further comprises a linkeramino sequence operably linked between the first amino acid sequence andthe second amino acid sequence.

31. The chimeric pesticidal polypeptide of embodiment 30, wherein thelinker amino acid sequence is selected from the group consisting of theamino acid sequences set forth in SEQ ID NOS: 28, 29, 30, and 31.

32. The chimeric pesticidal polypeptide of any one of embodiments 21-31,wherein the chimeric pesticidal polypeptide comprises increasedpesticidal activity against at least one pest, when compared to thepesticidal activity of the Cry endotoxin against the at least one pest.

33. The chimeric pesticidal polypeptide of embodiment 32, wherein the atleast one pest is an insect pest.

34. The chimeric pesticidal polypeptide of embodiment 33, wherein theinsect pest is an insect pest from the order Coleoptera or Lepidoptera.

35. The chimeric pesticidal polypeptide of embodiment 33, wherein theinsect pest is from the genus Diabrotica.

36. The chimeric pesticidal polypeptide of embodiment 33, wherein theinsect pest is western corn rootworm.

37. The chimeric pesticidal polypeptide of embodiment 33, wherein theinsect pest is black cutworm.

38. A nucleic acid molecule comprising a nucleotide sequence encoding achimeric pesticidal polypeptide, the chimeric pesticidal polypeptidecomprising a first amino acid sequence of a solubility-enhancingpolypeptide operably linked to a second amino acid sequence of a Cryendotoxin.

39. The nucleic acid molecule of embodiment 38, wherein the nucleotidesequence comprises a nucleotide sequence selected from the groupconsisting of:

-   -   (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17,        20, 22, 24, 26 or 32;    -   (b) a nucleotide sequence encoding the amino acid sequence set        forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;    -   (c) a nucleotide sequence comprising at least 80% nucleotide        sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32,        wherein the nucleotide sequence encodes a polypeptide having        pesticidal activity; and    -   (e) a nucleotide sequence encoding an amino acid sequence        comprising at least 80% amino acid sequence identity to the        amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23,        25, 27 or 33, wherein the nucleotide sequence encodes a        polypeptide having pesticidal activity.

40. The nucleic acid molecule of embodiment 38 or 39, wherein thesolubility-enhancing polypeptide is a MBP.

41. The nucleic acid molecule of embodiment 40, wherein the MBP isselected from the group consisting of MBPs having an accession numberset forth in Table 1.

42. The nucleic acid molecule of embodiment 38 or 39, wherein thesolubility-enhancing protein is a NusA.

43. The nucleic acid molecule of embodiment 38 or 39, wherein thesolubility-enhancing protein is a thioredoxin.

44. The nucleic acid molecule of embodiment 38 or 39, wherein the firstamino acid sequence comprises an amino acid sequence selected from thegroup consisting of the amino acid sequences set forth in SEQ ID NOS: 4,6, 34, 35 and 36.

45. The nucleic acid molecule of any one of embodiments 38-44, whereinthe Cry endotoxin is selected from the group consisting of Cryendotoxins set forth in Table 2.

46. The nucleic acid molecule of any one of embodiments 38-44, whereinthe second amino acid sequence encodes a Cry endotoxin selected from thegroup consisting of Cry endotoxins set forth in Table 2.

47. The nucleic acid molecule of embodiment 38, wherein the second aminoacid sequence comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 8, 10, 12 and 14.

48. The nucleic acid molecule of any one of embodiments 38-47, whereinthe chimeric pesticidal polypeptide further comprises a linker aminosequence operably linked between the first amino acid sequence and thesecond amino acid sequence.

49. The nucleic acid molecule of embodiment 48, wherein the linker aminoacid sequence is selected from the group consisting of the amino acidsequences set forth in SEQ ID NOS: 28, 29, 30, and 31.

50. The nucleic acid molecule of any one of embodiments 38-49, whereinthe chimeric pesticidal polypeptide comprises increased pesticidalactivity against at least one pest, when compared to the pesticidalactivity of the Cry endotoxin against the at least one pest.

51. The nucleic acid molecule of any one of embodiments 38-50, whereinthe at least one pest is an insect pest.

52. The nucleic acid molecule of embodiment 51, wherein the insect pestis an insect pest from the order Coleoptera or Lepidoptera.

53. The nucleic acid molecule of embodiment 51, wherein the insect pestis from the genus Diabrotica.

54. The nucleic acid molecule of embodiment 51, wherein the insect pestis western corn rootworm.

55. The nucleic acid molecule of embodiment 51, wherein the insect pestis black cutworm.

56. An expression cassette comprising a promoter that drives expressionin a host cell operably linked to a nucleic acid molecule of any one ofembodiments 38-55.

57. The expression cassette of embodiment 56, wherein the host cell is aplant cell.

58. The expression cassette of embodiment 57, wherein the promoter isselected from the group consisting of a chemical-inducible promoter,constitutive promoter, pest-inducible promoter, tissue-specific promoterand wound-inducible promoter.

59. The expression cassette of embodiment 58, wherein thetissue-specific promoter is selected from the group consisting of aleaf-preferred promoter, root-preferred promoter, seed-preferredpromoter, stalk-preferred promoter and vascular tissue-preferredpromoter.

60. A vector comprising the expression cassette of any one ofembodiments 56-59.

61. A transformed plant, plant part, plant cell or seed comprising inits genome the expression cassette of any one of embodiments 56-60.

62. The transformed plant, plant part or plant host cell of embodiment61, wherein the nucleotide sequence is stably incorporated into thegenome of the transformed plant, plant part, plant cell or seed.

63. A pesticidal composition comprising an effective amount of achimeric pesticidal polypeptide of any one of embodiments 21-37 or anactive variant or fragment thereof having pesticidal activity.

64. The pesticidal composition of embodiment 63, further comprisingbacteria expressing a nucleotide sequence encoding the chimericpesticidal polypeptide or a biologically active fragment or variantthereof.

65. The pesticidal composition of embodiment 63, further comprising atleast one agricultural protectant selected from the group consisting ofan acaricide, bactericide, fertilizer or micronutrient donor, fungicide,insecticide, nematocide and semiochemical.

66. The pesticidal composition of embodiment 65, wherein thesemiochemical is selected from the group consisting of an allomone,attractant, feeding pheromone, kairomone, repellent and stimulant.

67. A method of protecting a plant from an insect pest, the methodcomprising providing an effective amount of a pesticidal composition ofany one of embodiments 63-66 to reduce insect pest-related damage to theplant.

68. The method of embodiment 67, wherein the pesticidal composition isapplied by a procedure selected from the group consisting of spraying,dusting, broadcasting and seed coating.

69. The method of embodiment 67 or 68, wherein the chimeric pesticidalpolypeptide has pesticidal activity against an insect pest in the orderColeoptera, an insect pest in the order Lepiedoptera or both.

70. The method of any one of embodiments 67-69, wherein the insect pestis selected from the group consisting of species in the genusDiabrotica.

71. A plant comprising a polynucleotide construct stably incorporated inits genome, the polynucleotide construct comprising a nucleotidesequence operably linked to a promoter that drives expression in theplant, wherein the nucleotide sequence encodes a chimeric pesticidalpolypeptide comprising a first amino acid sequence of asolubility-enhancing polypeptide operably linked to a second amino acidsequence of a Cry endotoxin.

72. The plant of embodiment 71, wherein the nucleotide sequence isselected from the group consisting of:

-   -   (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17,        20, 22, 24, 26 or 32;    -   (b) a nucleotide sequence encoding the amino acid sequence set        forth in SEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33;    -   (c) a nucleotide sequence comprising at least 80% nucleotide        sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32,        wherein the nucleotide sequence encodes a polypeptide having        pesticidal activity; and    -   (e) a nucleotide sequence encoding an amino acid sequence        comprising at least 80% amino acid sequence identity to the        amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23,        25, 27 or 33, wherein the nucleotide sequence encodes a        polypeptide having pesticidal activity.

73. The plant of embodiment 71 or 72, wherein the solubility-enhancingpolypeptide is MBP.

74. The plant of embodiment 73, wherein the MBP is selected from thegroup consisting of MBPs having an accession number set forth in Table1.

75. The plant of embodiment 71 or 72, wherein the solubility-enhancingprotein is NusA.

76. The plant of embodiment 71 or 72, wherein the solubility-enhancingprotein is thioredoxin.

77. The plant of embodiment 71, wherein the first amino acid sequencecomprises an amino acid sequence selected from the group consisting ofthe amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

78. The plant of any one of embodiments 71-77, wherein the Cry endotoxinis selected from the group consisting of Cry endotoxins set forth inTable 2.

79. The plant of any one of embodiments 71-78, wherein the second aminoacid sequence encodes a Cry endotoxin selected from the group consistingof Cry endotoxins set forth in Table 2.

80. The plant of embodiment 71 or 72, wherein the second amino acidsequence comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 8, 10, 12 and 14.

81. The plant of any one of embodiments 71-80, wherein the chimericpesticidal polypeptide further comprises a linker amino sequenceoperably linked between the first amino acid sequence and the secondamino acid sequence.

82. The nucleic acid molecule of embodiment 81, wherein the linker aminoacid sequence is selected from the group consisting of the amino acidsequences set forth in SEQ ID NOS: 28, 29, 30 and 31.

83. The plant of any one of embodiments 71-82, wherein the chimericpesticidal polypeptide comprises increased pesticidal activity againstat least one pest, when compared to the pesticidal activity of the Cryendotoxin against the at least one pest.

84. The plant of embodiment 83, wherein the at least one pest is aninsect pest.

85. The plant of embodiment 84, wherein the insect pest is an insectpest from the order Coleoptera or Lepidoptera.

86. The plant of embodiment 84, wherein the insect pest is from thegenus Diabrotica.

87. The plant of embodiment 84, wherein the insect pest is western cornrootworm.

88. The plant of embodiment 84, wherein the insect pest is blackcutworm.

89. The plant of any one of embodiments 71-88, wherein the plant is amonocot.

90. The plant of embodiment 89, wherein the monocot is barley, maize,rice, rye, sorghum, sugarcane or wheat.

91. The plant of any one of embodiments 71-88, wherein the plant is adicot.

92. The plant of embodiment 91, wherein the dicot is alfalfa, Brassica,cotton, soybean or sunflower.

93. The plant of any one of embodiment 71-92, wherein the plant is aseed.

94. A method of protecting a plant, plant part or plant host cell froman insect pest, the method comprising the steps of:

-   -   (a) introducing into the plant, plant part or plant host cell an        expression cassette of any one of embodiments 56-59; and    -   (b) regenerating the plant, plant part or plant host cell into a        morphologically normal fertile plant, wherein the plant or part        thereof comprises a chimeric pesticidal polypeptide.

95. The method of embodiment 94, wherein the chimeric pesticidalpolypeptide has pesticidal activity against an insect pest in the orderColeoptera, an insect pest in the order Lepidoptera or both.

96. The method of embodiment 94, wherein the insect pest is selectedfrom the group consisting of species in the genus Diabrotica.

97. A method of enhancing the resistance of a plant to at least onepest, the method comprising introducing into a plant or at least oneplant cell a polynucleotide construct comprising a nucleotide sequenceoperably linked to a promoter that drives expression in the plant,wherein the nucleotide sequence encodes a chimeric pesticidalpolypeptide comprising a first amino acid sequence of asolubility-enhancing polypeptide operably linked to a second amino acidsequence of a Cry endotoxin.

98. The method of embodiment 97, wherein the nucleotide sequencecomprises a nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence set forth in SEQ ID NO: 1, 15, 17,        20, 22, 24, 26 or 32;    -   (b) a nucleotide sequence encoding an amino acid sequence        comprising the amino acid sequence set forth in SEQ ID NO: 2,        16, 18, 21, 23, 25, 27 or 33;    -   (c) a nucleotide sequence comprising at least 80% nucleotide        sequence identity to SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32,        wherein the nucleotide sequence encodes a polypeptide having        pesticidal activity; and    -   (e) a nucleotide sequence encoding an amino acid sequence        comprising at least 80% amino acid sequence identity to the        amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23,        25, 27 or 33, wherein the nucleotide sequence encodes a        polypeptide having pesticidal activity.

99. The method of embodiment 97 or 98, wherein solubility-enhancingpolypeptide is MBP.

100. The method of embodiment 99, wherein the MBP is selected from thegroup consisting of MBPs having an accession number set forth in Table1.

101. The method of embodiment 97 or 98, wherein the solubility-enhancingprotein is NusA.

102. The method of embodiment 97 or 98, wherein the solubility-enhancingprotein is thioredoxin.

103. The method of embodiment 97, wherein the first amino acid sequencecomprises an amino acid sequence selected from the group consisting ofthe amino acid sequences set forth in SEQ ID NOS: 4, 6, 34, 35 and 36.

104. The method of any one of embodiments 97-103, wherein the Cryendotoxin is selected from the group consisting of Cry endotoxins setforth in Table 2.

105. The method of any one of embodiments 97-104, wherein the secondamino acid sequence comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 8, 10, 12 and 14.

106. The method of any one of embodiments 97-105, wherein the chimericpesticidal polypeptide further comprises a linker amino sequenceoperably linked between the first amino acid sequence and the secondamino acid sequence.

107. The method of embodiment 106, wherein the linker amino acidsequence is selected from the group consisting of the amino acidsequences set forth in SEQ ID NOS: 28, 29, 30 and 31.

108. The method of any one of embodiments 97-107, wherein the chimericpesticidal polypeptide comprises increased pesticidal activity againstat least one pest, when compared to the pesticidal activity of the Cryendotoxin against the at least one pest.

109. The method of any one of embodiments 97-108, further comprisingregenerating a plant comprising the polynucleotide construct.

110. The method of any one of embodiments 97-109, wherein the plant hasenhanced resistance to at least one pest, when compared to a controlplant lacking the polynucleotide construct.

111. The method of embodiment 110, wherein the at least one pest is aninsect pest.

112. The method of embodiment 111, wherein the insect pest is an insectpest from the order Coleoptera or Lepidoptera.

113. The method of embodiment 111, wherein the insect pest is from thegenus Diabrotica.

114. The method of embodiment 111, wherein the insect pest is westerncorn rootworm.

115. The method of embodiment 111, wherein the insect pest is blackcutworm.

116. The method of any one of embodiments 97-115, wherein the plant is amonocot.

117. The method of embodiment 116, wherein the monocot is barley, maize,rice, rye, sorghum, sugarcane or wheat.

118. The method of any one of embodiments 97-115, wherein the plant is adicot.

119. The method of embodiment 118, wherein the dicot is alfalfa,Brassica, cotton, soybean or sunflower.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 depicts the structure of E. coli maltose-binding protein (MBP)-BtCry protein fusion of the present invention.

=maltose binding protein

=Bt Cry protein.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention is based on the discovery that pesticidal activityof Cry endotoxins can be enhanced by operably linking asolubility-enhancing polypeptide, such as, for example, amaltose-binding protein (MBP), to a Cry endotoxin or insecticidallyactive fragment thereof. Moreover, insect pest resistance can beovercome by operably linking an MBP to a Cry endotoxin by the methodsdisclosed herein. The present invention therefore provides compositionsand methods for enhancing Cry endotoxin activity, for overcoming plantpest resistance, and for protecting plants from pests, especially insectpests.

While the invention does not depend on a particular biologicalmechanism, the fusion of a solubility-enhancing polypeptide to a Cryendotoxin or insecticidally active fragment thereof may act to raise thesolubility of the Cry protein, particularly in the insect gutenvironment, more particularly in the gut environment of a Coleopteraninsect. That is the solubility of the fusion protein has a highersolubility at a slightly acidic pH than the Cry protein has at the samepH. In a preferred embodiment of the invention, with activated Cyr toxinthat is used in making the fusion protein of the invention has anisoelectric point (pI) from about pH 7 to about pH 8. It is recognizedthat in the digestive system in Lepidopteran insects, pH is relativelyhigh. In such alkaline conditions, an activated Bt Cry toxin typicallyis soluble but in the slightly acidic pH of the Coleopteran gutenvironment in which pH is slightly acidic, solubility of the activatedBt Cry toxin can be much lower.

Compositions and methods are provided for enhancing the pesticidalactivity of Cry endotoxins by operably linking a solubility-enhancingprotein to a Cry endotoxin and for protecting plants from insect pests.The compositions and methods disclosed herein include recombinantnucleic acid molecules that encode chimeric pesticidal polypeptides,expression cassettes and other nucleotide constructs including thenucleic acid molecules described herein organisms transformed with thenucleic acid molecules described herein, isolated chimeric pesticidalpolypeptides, and pesticidal compositions having the chimeric pesticidalpolypeptides, as well as methods of using the same. The compositions andmethods therefore find use in enhancing pesticidal activity ofpesticidal proteins and in protecting plants from insect pests.

As used herein, a “solubility-enhancing polypeptide” is any polypeptidethat when operably linked to a Cry endotoxin or insecticidally activefragment thereof enhances the pesticidal activity of the Cry endotoxinor insecticidally active fragment against at least one insect pest,preferably a Coleopteran insect pest, when compared to the pesticidalactivity of the same Cry endotoxin or insecticidally active fragmentthereof in the absence of an operably linked solubility-enhancingpolypeptide. In certain embodiments, the solubility enhancingpolypeptides of the present invention are capable of increasing thesolubility of an operable linked Cry endotoxin or insecticidally activefragment thereof at a slightly acidic pH that is found, for example, inthe gut environment of a Coleopteran insect. Preferably, a chimericpesticidal polypeptide of the present invention, which comprises asolubility-enhancing polypeptide of the present invention operablylinked to a Cry endotoxin or insecticidally active fragment thereof,comprises an increased solubility in an environment that has a slightlyacidic pH such as, for example, the gut environment of a Coleopteraninsect, than the same Cry endotoxin or insecticidally active fragmentthereof lacking the operably linked solubility-enhancing polypeptide.

As used herein, “pest” or “plant pest” means an organism that interfereswith or is harmful to plant development and/or growth. Such plant pestsinclude, but are not limited to, nematodes, insect, viruses, viroids,mites, fungal pathogens, bacteria and any other plant pests disclosedherein. Accordingly, the polynucleotides and polypeptides of theinvention can be used to enhance resistance of plants to plant pests.One of skill in the art, however, understands that not all polypeptidesare equally effective against all plant pests. The chimeric pesticidalpolypeptides described herein display activity against plant pests suchas insect pests, which may include economically important agronomic,forest, greenhouse, nursery ornamentals, food and fiber, public andanimal health, domestic and commercial structure, household and storedproduct pests. Therefore, of interest herein are chimeric pesticidalpolypeptides for use in protecting plants from insect pests.

As used herein, “pesticidal polypeptide” means a peptide, polypeptide orprotein that has biological activity against insect pests (i.e., ispesticidal).

As used herein, “pesticidal” or “pesticidal activity” means capable ofkilling insect pests. Likewise, “pesticidal” or “pesticidal activity”means capable of inhibiting insect growth. As such, the chimericpesticidal polypeptides described herein are capable of inhibitinggrowth or reproduction of or of killing, at least one plant pest,particularly at least one insect pest.

By “insecticidal activity” is intended the ability of a polypeptide ofthe invention or a composition comprising the polypeptide (or otheragent, chemical or composition) to inhibit the growth of or damage to aplant caused by, at least one insect pest.

As used herein, “enhance” and the like means increasing anactivity/effectiveness of a pesticidal polypeptide such as a Cryendotoxin against a particular pest by operably linking the Cryendotoxin to a solubility-enhancing polypeptide, where theactivity/effectiveness of the resulting chimeric pesticidal polypeptideis increased by about 1% to about 5%, about 5% to about 10%, about 10%to about 20%, about 20% to about 30%, about 30% to about 40%, about 40%to about 50%, about 50% to about 60%, about 60% to about 70%, about 70%to about 80%, about 80% to about 90% or about 90% to about 100% whencompared to a wild-type Cry endotoxin. Alternatively, theactivity/effectiveness of the chimeric pesticidal polypeptide isincreased by about 1%, about 2%, about 3%, about 4%, about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or morewhen compared to the wild-type Cry endotoxin. In addition, “enhance” canmean that the host pest range of the pesticidal polypeptide is expandedto include additional pests.

As used herein, “about” means within a statistically meaningful range ofa value such as a stated concentration, length, molecular weight,percentage, pH, time frame, temperature or volume. Such a value or rangecan be within an order of magnitude, typically within 20%, moretypically within 10%, and even more typically within 5% of a given valueor range. The allowable variation encompassed by “about” will dependupon the particular system under study, and can be readily appreciatedby one of skill in the art.

As used herein, “chimeric polypeptide” or “chimeric pesticidalpolypeptide” means a polypeptide having a first amino acid sequencederived from a first source operably linked, covalently ornon-covalently, to a second amino acid sequence derived from a secondsource, where the first and second source are not the same. A firstsource and a second source that are not the same can include twodifferent organisms or two different proteins from the same organism ora biological source and a synthetic source or even two differentsynthetic sources. A biological source can include any non-syntheticallyproduced nucleotide or amino acid sequence (e.g., a genomic or cDNAsequence, a plasmid or viral vector, a native virion or a mutant oranalog). A synthetic source can include a nucleotide or amino acidsequence produced chemically and not by a biological source (e.g., solidphase synthesis of amino acid sequences). The chimeric pesticidalpolypeptide can be produced by expressing a recombinant nucleic addmolecule encoding a polypeptide having at least two parts or can beproduced synthetically.

A chimeric pesticidal polypeptide of the present can further comprise,for example, a linker molecule (also referred to here as a “linker)between the first and second amino acid sequences. Examples of linkersthat can be used in the methods of the present invention are the NEBpMAL, SA, NusA, and TrxA linkers having the amino acid sequences setforth in SEQ ID NOS: 28, 29, 30, and 31, respectively.

Solubility-enhancing polypeptides of the present include, but are notlimited to, maltose-binding protein (MBP), thioredoxin (e.g., TrxA),transcription elongation factor NusA, glutathione-S-transferase (GST),mistic, small ubiquitin-related modifier (SUMO), protein disulfideisomerase DsbC, and thiol:disulfide interchange protein DsbD, andvariants and fragments thereof that when operably linked to a Cryendotoxin or insecticidally active fragment thereof enhance thepesticidal activity of an operably linked Cry endotoxin orinsecticidally active fragment against at least one insect pest,preferably a Coleopteran insect pest, when compared to the pesticidalactivity of the same Cry endotoxin or insecticidally active fragmentthereof in the absence of an operably linked solubility-enhancingpolypeptide.

As used herein, “maltose-binding protein” or “MBP” means a polypeptidethat is a member of the maltodextrin transport system that bindsmaltodextrins (e.g., maltose, maltotriose and trehalose) with micromolaraffinity and that is essential for an energy-dependent translocation ofmaltodextrins through a cytoplasmic membrane of some prokaryotes. See,Boos & Shuman (1998) Microbiol. Mol. Biol. Rev. 62:204-229. For example,in Escherichia coli, the maIE gene encodes a 396 amino acid residuepre-MBP, which subsequently is processed into MBP upon cleavage of a 26amino acid N-terminal signal peptide. See, e.g., Duplay et al. (1984) J.Biol. Chem. 259:10606-10613.

In one embodiment of the invention, the chimeric pesticidal polypeptidecomprises an amino acid sequence of an MBP operably linked to an aminoacid sequence of a Cry endotoxin including, for example, the chimericpesticidal polypeptides comprising the amino acid sequences set forth inSEQ ID NOS: 2, 21, 23, 25, 27, and 33 and encoded by the nucleotidesequences set forth in SEQ ID NOS: 1, 20, 22, 24, 26, and 32,respectively.

In another embodiment, the chimeric pesticidal polypeptide comprises anamino acid sequence of NusA operably linked to an amino acid sequence ofa Cry endotoxin including, for example, the chimeric pesticidalpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:16 and encoded by the nucleotide sequence set forth in SEQ ID NO: 15.

In yet another embodiment, the chimeric pesticidal polypeptide comprisesan amino acid sequence of thioredoxin operably linked to an amino acidsequence of a Cry endotoxin including, for example, the chimericpesticidal polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 18 and encoded by the nucleotide sequence set forth in SEQ IDNO: 17.

The present invention comprises the use of an solubility-enhancingpolypeptide that when fused to a Cry endotoxin polypeptide increases thepesticidial activity of against at least one pest, preferably an insectpest, more preferably an insect pest from the order Coleoptera orLepidoptera, even more preferably an insect pest from the genusDiabrotica, most preferably the western corn rootworm (Diabroticavirgifera virgifera). In certain embodiments, the solubility-enhancingpolypeptide selected from the group consisting of MBP, NusA, and TrxA.

The present invention does not depend on the use of a particularsolubility-enhancing polypeptide. Any solubility-enhancing polypeptidethat, when fused to a Cry endotoxin polypeptide, is capable increasingthe pesticidial activity of the Cry endotoxin polypeptide against atleast one pest can be used in the methods and compositions disclosedherein. Such solubility enhancing polypeptides include, for example,pre-proteins and mature proteins, and variants and fragments thereof. Incertain embodiments, the solubility-enhancing polypeptide is MBP. MBPsof the present invention include, but are not limited, full-length MBP(also referred to as “pre-MBP”, a mature form of an MBP, a fragment of afull-length or mature MBP or a variant. Unless expressed stated hereinor otherwise apparent form the context, the terms “maltose-bindingprotein” and “MBP” encompass such full-length and mature forms of MBPsas well as fragments and variants thereof.

The amino acid and/or nucleotide sequences for a number of MBPs havebeen disclosed and can be used in the compositions and methods of thepresent invention including, but not limited to, the following thoseMBPs having the following accession numbers in Table 1.

TABLE 1 GenBank Accession Number* NP_290668 ZP_03064501 CBG37227NP_709885 ACI74011 YP_002415175 YP_001726921 EFK20320 ZP_02999303YP_312946 ZP_06651607 ACC91724 NP_756856 ACF57854 ACF57853 ABO28850AAB86559 AAC83813 BAI57431 AAQ93661 ACI46135 ACI46133 AAK55118 AAB87675ZP_02904048 YP_002385139 ZP_06356417 YP_003367151 YP_002639794 NP_458527YP_001338045 ACY91424 ZP_03381315 YP_219095 AAX68014 AAL23053 NP_463094ZP_04558600 YP_002228802 ZP_02700756 YP_002149143 CAA38189 CBK84491ZP_06390929 YP_001455393 YP_003610806 YP_001174979 YP_001572421YP_002241024 P18815 YP_003212142 YP_001436222 YP_001480694 ZP_06712852ZP_04616043 ZP_04638283 ZP_06191619 ZP_04626936 ZP_04633703 ZP_04613648NP_667372 ZP_04641958 YP_001161578 YP_001008013 ZP_04625082 YP_003294268YP_002931694 ZP_06638363 BAE44434 YP_003743846 YP_003039205 NP_927811ZP_05729913 YP_003714385 YP_003469920 YP_003729949 YP_003520206YP_002891253 ZP_03366279 ZP_05920010 YP_003007381 ZP_06636620YP_001343631 YP_003255618 ZP_04976824 YP_089260 ZP_06157313 YP_002474876YP_001447347 ZP_01984309 ZP_04754507 ZP_06177188 NP_800911 ZP_06180148ZP_02477508 ZP_01258612 ZP_05878943 ZP_05629984 YP_001053928 YP_132094ZP_05882865 ZP_02194153 ZP_01221443 ZP_05927269 NP_763457 ZP_05943387ZP_01865449 ZP_05717553 ZP_06032225 ZP_06079586 ZP_05239461 YP_002812521NP_233329 ZP_01970977 ZP_04918366 ZP_05719582 ZP_05886194 ZP_05117778ZP_06941932 ZP_04415908 ZP_01950896 ZP_01676462 ZP_01897017 YP_001343632YP_132089 ZP_06938154 YP_003007380 ZP_03352864 ZP_01221449 ZP_00988394ZP_01811558 YP_856203 YP_001142458 NP_936112 ZP_01062901 NP_763137YP_206757 ZP_00988462 ZP_04403894 ZP_05120494 ZP_01947956 ZP_01062959ZP_01865441 YP_431775 ZP_01895948 YP_001173956 ZP_03375554 YP_001188617ZP_00135549 ZP_03699982 YP_003269532 YP_003548264 ZP_01893753ZP_01112736 YP_431868 ZP_01157584 ZP_01115171 ZP_03369842 ZP_01955972ZP_01955970 ZP_06938864 YP_002352611 YP_002250432 YP_002534909 NP_229009YP_001245135 YP_002534307 YP_001739017 YP_001244560 YP_003346096YP_002250571 CAA72193 NP_229635 YP_002352749 YP_001470540 NP_623418ZP_05493657 YP_001662811 YP_002941091 YP_003672376 YP_146557 ZP_04392773YP_003677351 ZP_05336993 YP_003477456 AAY89718 YP_001568299 YP_001410291ZP_03385039 YP_001568464 YP_001124738 YP_604964 YP_003514612 ZP_03146818ZP_04152680 ZP_04158384 YP_001305848 YP_001142178 ZP_04307633YP_002509363 ZP_06220504 ZP_04121933 ZP_04204735 ZP_03232052YP_003148220 ZP_04086064 ZP_04258265 ZP_04296475 ZP_02397670 ZP_00236932ZP_04187645 ZP_03105903 ZP_03238270 YP_896372 YP_038073 YP_085351NP_846464 ZP_04128078 ZP_04285672 YP_003186266 ZP_02948002 YP_002447583CAB65651 NP_980358 ZP_04302212 ZP_03493519 ZP_02878398 ZP_04176050ZP_05183404 ZP_04290906 ZP_04199019 ZP_04263630 YP_001646638 ZP_04229423ZP_04170363 YP_002334500 BAB40635 ZP_04218748 ZP_02866107 YP_697032YP_003258733 ZP_02953587 YP_177014 YP_051264 ZP_02638879 YP_003018517ZP_04607307 ZP_03828721 YP_003116555 ZP_03832162 YP_002316535 NP_294284NP_563259 YP_002785582 YP_002886868 ZP_06681977 ZP_05667047 ZP_05664221ZP_03981553 ZP_06699376 YP_001488817 YP_001375943 ZP_00603967ZP_05132228 YP_699603 YP_002574412 YP_001814130 YP_003445304 ZP_04878976ZP_03055079 ZP_01861955 YP_002918149 YP_002240053 YP_001334109YP_001813248 ZP_06549508 ZP_06329195 ZP_04864724 YP_001179261 YP_144918YP_039671 NP_645005 NP_370738 YP_003465347 ZP_05877552 ZP_04016117YP_001439475 BAI87082 ADI96692 CBI48099 YP_850342 YP_005257 ZP_00233442YP_003613139 ZP_06315382 YP_946709 NP_391341 AAD42742 EFK15520ZP_05300115 ZP_05298949 YP_001695468 ZP_02185856 YP_830242 YP_288893NP_471563 YP_003208908 YP_002958966 YP_001836789 ZP_01827852 YP_415662NP_359509 BAB18102 YP_002739191 YP_003154662 ZP_03497002 YP_003696753YP_003684577 ZP_07053052 ZP_05231671 ZP_02711433 ZP_01821820 ZP_01821214NP_346527 ZP_01408156 ZP_02186034 BAC10980 YP_003336687 NP_670540YP_071605 ZP_06640983 YP_003723548 ZP_04853514 YP_002743432 YP_001399898YP_077886 YP_002038696 NP_391296 ZP_06611384 ZP_06198009 YP_003339007YP_003729851 ZP_03958786 ZP_01170933 YP_081353 YP_001719722 ZP_06059512YP_001449426 ZP_06872846 ZP_04851795 YP_003241054 YP_002473273YP_001396549 YP_920853 ZP_06190079 BAI87023 YP_001664668 YP_003704868ZP_06899649 NP_693481 YP_003314160 YP_003334475 ZP_05647136 YP_003203952YP_056254 ZP_06014674 ZP_05687198 YP_003506902 ZP_04449975 YP_002958266YP_002948859 YP_003677653 ZP_06428097 YP_003781558 ZP_03925962YP_001199499 NP_579667 YP_003003618 YP_003678645 ZP_03225712 ZP_01460386YP_003326738 YP_002744243 YP_002746719 YP_002123617 AAA26922YP_003477807 ZP_02329884 ZP_04431587 ZP_01461944 NP_242885 YP_003161478ZP_06533915 YP_001137791 YP_003009537 NP_125870 YP_002308182 ZP_06885100ZP_04876799 YP_001559408 YP_003699585 ZP_01130620 YP_184184 ZP_05621839NP_243792 YP_002883083 YP_003637265 YP_003425427 YP_003061750YP_002997014 ZP_07076764 ZP_05749331 YP_602654 NP_269430 NP_737480ZP_06808450 YP_003009727 YP_002565304 YP_598732 YP_280516 YP_002881503NP_784007 YP_002246441 ZP_02045060 YP_002509787 ZP_06608272 YP_002136599ZP_06363693 EFK02214 ZP_05431814 YP_136875 ZP_06662656 YP_001880661YP_177522 ZP_06048003 YP_002387348 ZP_01983428 YP_003229678 ZP_04402673YP_467337 ZP_06365288 CAL69747 YP_002881799 ZP_04412511 ZP_01951248 *Theamino acid and corresponding nucleotide sequences of the accessionnumbers in Table 1 are herein incorporated by reference.

In certain embodiments of the invention, the MBP comprises the aminoacid sequence set forth in SEQ ID NO: 4 or 6 or fragment or variantthereof. Any nucleotide sequence encoding the amino acid sequence setforth in SEQ ID NO: 4 or 6 or fragment or variant thereof, can be usedin the methods and compositions of the present invention. In someembodiments of the invention, the nucleotide sequences of the inventionwill be optimized for expression in a host organism or cell of interest,particularly a plant, more particularly a crop plant, most particularlya maize plant.

As used herein, “Cry endotoxin” means a δ-endotoxin encoded by cry(crystal protein) genes that are located mainly on large plasmids inmembers of the genus Bacillus, although chromosomally encoded Cryendotoxins have been reported. See, Ben-Dov et al. (1996) Appl. Environ.Microbiol. 62:3140-3145; Berry et al. (2002) Appl. Environ. Microbiol.68:5082-5095; Gonzáles et al. (1981) Plasmid 5:351-365; Lereclus et al.(1982) Mol. Gen. Genet. 186:391-398; and Trisrisook et al. (1990) Appl.Environ. Microbiol. 56:1710-1716. Cry endotoxins do not have a broadspectrum of activity, so they typically do not kill beneficial insects.Furthermore, Cry endotoxins are non-toxic to mammals, including humans,domesticated animals and wildlife.

Cry endotoxins generally have five conserved sequence domains, and threeconserved structural domains. See, e.g., de Maagd et al. (2001) TrendsGenetics 17:193-199. The first conserved structural domain (Domain I)consists of seven alpha helices and is involved in membrane insertionand pore formation. The second conserved structural domain (Domain II)consists of three beta-sheets arranged in a Greek key configuration, andthe third conserved structural domain (Domain III) consists of twoantiparallel beta-sheets in “jelly-roll” formation. Domains II and IIIare involved in receptor recognition and binding, and are thereforeconsidered determinants of toxin specificity.

As used herein, “insect pest” means an organism in the phylum Arthropodathat interferes with or is harmful to plant development and/or growth,and more specifically means an organism in the class Insecta. The classInsecta can be divided into two groups historically treated assubclasses: (1) wingless insects, known as Apterygota; and (2) wingedinsects, known as Pterygota. The insect pests can be adults, larvae oreven ova. A preferred developmental stage for testing for pesticidalactivity is larvae or other immature form of the insect pest. Methods ofrearing insect larvae and performing bioassays are well known in theart. See, e.g., Czapla & Lang (1990) J. Econ. Entomol. 83:2480-2485;Griffith & Smith (1977) J. Aust. Ent. Soc. 16:366; and Keiper & Foote(1996) Hydrobiologia 339:137-139; as well as U.S. Pat. No. 5,351,643.For example, insect pests can be reared in total darkness at about 20°C. to about 30° C. and from about 30% to about 70% relative humidity.

Compositions comprising chimeric pesticidal polypeptide-encoding nucleicacid molecules and chimeric pesticidal polypeptides also are provided.The compositions include chimeric pesticidal polypeptides that comprisea solubility-enhancing polypeptide fused to a Cry endotoxin orbiologically active fragment thereof. Nucleotide sequences that encodethe chimeric pesticidal polypeptides can be derived from the chimericpesticidal polypeptide sequence and produced using any method known inthe art. The compositions also include variants and fragments of thechimeric pesticidal-encoding nucleic acid molecules and chimericpesticidal polypeptides. The isolated, chimeric pesticidal-encodingnucleic acid molecules can be used to create transgenic organisms, suchas plants, that are resistant to an insect pest susceptible to thepesticidal polypeptide.

The presently disclosed methods and compositions provides for chimericpesticidal polypeptides with improved efficacy and nucleic acidmolecules encoding the chimeric pesticidal polypeptides. The chimericpesticidal polypeptides comprise solubility-enhancing polypeptide fused(i.e., operably linked) to a Cry endotoxin or biologically activefragment thereof and have enhanced pesticidal activity against at leastone plant pest, when compared to the pesticidal activity of the Cryendotoxin or biologically active fragment thereof that has not beenfused to a solubility-enhancing polypeptide.

As used herein, “pesticide,” “pesticidal polypeptide,” or “pesticidalprotein” mean a polypeptide that is capable of killing the pest orinhibiting its growth, feeding or reproduction. One of skill in the artunderstands that not all substances or mixtures thereof are equallyeffective against all pests. “Chimeric pesticidal polypeptides” or“chimeric pesticidal proteins” are “pesticidal polypeptides” or“pesticidal proteins”, to which a solubility-enhancing polypeptide hasbeen fused as disclosed herein. Of particular interest herein arepesticidal polypeptides and chimeric pesticidal polypeptides that act asinsecticides and thus have biological activity against insect pests.

As used herein, “pest” means an organism that interferes with or isharmful to plant development and/or growth. Examples of pests include,but are not limited to, algae, arachnids (e.g., acarids including mitesand ticks), bacteria (e.g., plant pathogens including Xanthomonas spp.and Pseudomonas spp.), crustaceans (e.g., pillbugs and sowbugs); fungi(e.g., members in the phylum Ascomycetes or Basidiomycetes, andfungal-like organisms including Oomycetes such as Pythium spp. andPhytophthora spp.), insects, mollusks (e.g., snails and slugs),nematodes (e.g., soil-transmitted nematodes including Clonorchis spp.,Fasciola spp., Heterodera spp., Globodera spp., Opisthorchis spp. andParagonimus spp.), protozoans (e.g., Phytomonas spp.), viruses (e.g.,Comovirus spp., Cucumovirus spp., Cytorhabdovirus spp., Luteovirus spp.,Nepovirus spp., Potyvirus spp., Tobamovirus spp., Tombusvirus spp. andTospovirus spp.), viroids, parasitic plants, and weeds.

Of particular interest herein are insect pests. As used herein, “insectpest” means an organism in the phylum Arthropoda that interferes with oris harmful to plant development and/or growth, and more specificallymeans an organism in the class Insecta. The class Insecta can be dividedinto two groups historically treated as subclasses: (1) winglessinsects, known as Apterygota; and (2) winged insects, known asPterygota. Examples of insect pests include, but are not limited to,insects in the orders Coleoptera, Diptera, Hemiptera, Homoptera,Hymenoptera, Isoptera, Lepidoptera, Mallophaga orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichopteraand Thysanura, particularly Coleoptera and Lepidoptera. Whiletechnically not insects, arthropods such as arachnids, especially in theorder Acari, are included in “insect pest.” Insect pests includeeconomically important agronomic, forest, greenhouse, nurseryornamentals, food and fiber, public and animal health, domestic andcommercial structure, household, and stored product pests.

Insects of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the family NoctuidaeAgrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison(western cutworm); A. segetum Denis & Schiffermüller (turnip moth); A.subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner(cotton leaf worm); Anticarsia gemmatalis Hübner (velvetbeancaterpillar); Athetis mindara Barnes and McDunnough (rough skinnedcutworm); Earias insulana Boisduval (spiny bollworm); E. vittellaFabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citruscutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpaarmigera Hübner (American bollworm); H. zea Boddie (corn earworm orcotton bollworm); Heliothis virescens Fabricius (tobacco budworm);Hypena scabra Fabricius (green cloverworm); Hyponeuma taltula Schaus;(Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Melanchra picta Harris (zebra caterpillar); Mocislatipes Guenée (small mocis moth); Pseudaletia unipuncta Haworth(armyworm); Pseudoplusia includens Walker (soybean looper); Richiaalbicosta Smith (Western bean cutworm); Spodoptera frugiperda J E Smith(fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius(tobacco cutworm, cluster caterpillar); Trichoplusia ni Hübner (cabbagelooper); borers, casebearers, webworms, coneworms, and skeletonizersfrom the families Pyralidae and Crambidae such as Achroia grisellaFabricius (lesser wax moth); Amyelois transitella Walker (navalorangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth);Cadra cautella Walker (almond moth); Chilo partellus Swinhoe (spottedstalk borer); C. suppressalis Walker (striped stem/rice borer); C.terrenellus Pagenstecher (sugarcane stemp borer); Corcyra cephalonicaStainton (rice moth); Crambus caliginosellus Clemens (corn rootwebworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocismedinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grapeleaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalisStoll (pickleworm); Diatraea flavipennella Box; D. grandiosella Dyar(southwestern corn borer), D. saccharalis Fabricius (sugarcane borer);Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Eoreumaloftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco(cacao) moth); Galleria mellonella Linnaeus (greater wax moth);Hedylepta accepta Butler (sugarcane leafroller); Herpetogrammalicarsisalis Walker (sod webworm); Homoeosoma electellum Hulst(sunflower moth); Loxostege sticticalis Linnaeus (beet webworm); Marucatestulalis Geyer (bean pod borer); Orthaga thyrisalis Walker (tea treeweb moth); Ostrinia nubilalis Hübner (European corn borer); Plodiainterpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker(yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms, and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Adoxophyes oranaFischer von Rösslerstamm (summer fruit tortrix moth); Archips spp.including A. argyrospila Walker (fruit tree leaf roller) and A. rosanaLinnaeus (European leaf roller); Argyrotaenia spp.; Bonagota salubricolaMeyrick (Brazilian apple leafroller); Choristoneura spp.; Cochylishospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham(filbertworm); C. pomonella Linnaeus (codling moth); Endopiza viteanaClemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth);Grapholita molesta Busck (oriental fruit moth); Lobesia botrana Denis &Schiffermüller (European grape vine moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); andSuleima helianthana Riley (sunflower bud moth).

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakSilkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Erechthias flavistriata Walsingham (sugarcane bud moth);Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americanaGuérin-Méneville (grapeleaf skeletonizer); Heliothis subflexa Guenée;Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury(fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm);Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L.fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicisLinnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth);Malacosoma spp.; Manduca quinquemaculata Haworth (five spotted hawkmoth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobaccohornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp.;Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer(giant swallowtail orange dog); Phryganidia californica Packard(California oakworm); Phyllocnistis citrella Stainton (citrusleafminer); Phyllonorycter blancardella Fabricius (spotted tentiformleafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapaeLinnaeus (small white butterfly); P. napi Linnaeus (green veined whitebutterfly); Platyptilia carduidactyla Riley (artichoke plume moth);Plutella xylostella Linnaeus (diamondback moth); Pectinophoragossypiella Saunders (pink bollworm); Pontia protodice Boisduval &Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorouslooper); Schizura concinna J. E. Smith (red humped caterpillar);Sitotroga cerealella Olivier (Angoumois grain moth); Telchin licus Drury(giant sugarcane borer); Thaumetopoea pityocampa Schiffermüller (pineprocessionary caterpillar); Tineola bisselliella Hummel (webbingclothesmoth); Tuta absoluta Meyrick (tomato leafminer) and Yponomeutapadella Linnaeus (ermine moth).

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae, and Curculionidaeincluding, but not limited to: Anthonomus grandis Boheman (boll weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepesabbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius(clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice waterweevil); Metamasius hemipterus hemipterus Linnaeus (West Indian caneweevil); M. hemipterus sericeus Olivier (silky cane weevil); Sitophilusgranarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil);Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidusLeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden(maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelusobscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumberbeetles, rootworms, leaf beetles, potato beetles, and leafminers in thefamily Chrysomelidae including, but not limited to: Chaetocnema ectypaHorn (desert corn flea beetle); C. pulicaria Melsheimer (corn fleabeetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberiSmith & Lawrence (northern corn rootworm); D. undecimpunctata howardiBarber (southern corn rootworm); D. virgifera virgifera LeConte (westerncorn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle);Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferaeGoeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflowerbeetle); beetles from the family Coccinellidae including, but notlimited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafersand other beetles from the family Scarabaeidae including, but notlimited to: Antitrogus parvulus Britton (Childers cane grub);Cyclocephala borealis Arrow (northern masked chafer, white grub); C.immaculata Olivier (southern masked chafer, white grub); Dermolepidaalbohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugicepsLeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French's canegrub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicusBlatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub);P. latifrons LeConte (June beetle); Popillia japonica Newman (Japanesebeetle); Rhizotrogus majalis Razoumowsky (European chafer); carpetbeetles from the family Dermestidae; wireworms from the familyElateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal(wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Cteniceraspp.; Aeolus spp.; bark beetles from the family Scolytidae; beetles fromthe family Tenebrionidae; beetles from the family Cerambycidae such as,but not limited to, Migdolus fryanus Westwood (longhorn beetle); andbeetles from the Buprestidae family including, but not limited to,Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestidbeetle).

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicomis Loew (corn blotch leafminer); midgesincluding, but not limited to: Contarinia sorghicola Coquillett (sorghummidge); Mayetiola destructor Say (Hessian fly); Neolasiopteramurtfeldtiana Felt, (sunflower seed midge); Sitodiplosis mosellana Géhin(wheat midge); fruit flies (Tephritidae), Oscinella frit Linnaeus (fritflies); maggots including, but not limited to: Delia spp. includingDelia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulbfly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser houseflies); Meromyza americana Fitch (wheat stem maggot); Musca domesticaLinnaeus (house flies); Stomoxys calcitrans Linnaeus (stable flies));face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; andother muscoid fly pests, horse flies Tabanus spp.; bot fliesGastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer fliesChrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera,mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black fliesProsimulium spp.; Simulium spp.; biting midges, sand flies, sciarids,and other Nematocera.

Included as insects of interest are those of the order Hemiptera suchas, but not limited to, the following families: Adelgidae, Aleyrodidae,Aphididae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae,Cixiidae, Coccidae, Coreidae, Dactylopiidae, Delphacidae, Diaspididae,Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae,Membracidae, Miridae ortheziidae, Pentatomidae, Phoenicococcidae,Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

Agronomically important members from the order Hemiptera include, butare not limited to: Acrosternum hilare Say (green stink bug);Acyrthisiphon pisum Harris (pea aphid); Adelges spp. (adelgids);Adelphocoris rapidus Say (rapid plant bug); Anasa tristis De Geer(squash bug); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli(black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A.maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A.spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner(sugarcane scale); Aulacorthum solani Kaltenbach (foxglove aphid);Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B.argentifolii Bellows & Perring (silverleaf whitefly); Blissusleucopterus leucopterus Say (chinch bug); Blostomatidae spp.;Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricolaFoerster (pear psylla); Calocoris norvegicus Gmelin (potato capsid bug);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae spp.;Coreidae spp.; Corythuca gossypii Fabricius (cotton lace bug);Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant (suckfly);Deois flavopicta St

l (spittlebug); Dialeurodes citri Ashmead (citrus whitefly);Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Duplachionaspis divergensGreen (armored scale); Dysaphis plantaginea Paaserini (rosy appleaphid); Dysdercus suturellus Herrich-Schäffer (cotton stainer);Dysmicoccus boninsis Kuwana (gray sugarcane mealybug); Empoasca fabaeHarris (potato leafhopper); Eriosoma lanigerum Hausmann (woolly appleaphid); Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir(Island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say(brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stinkbug); Graptostethus spp. (complex of seed bugs); and Hyalopterus pruniGeoffroy (mealy plum aphid); Icerya purchasi Maskell (cottony cushionscale); Labopidicola allii Knight (onion plant bug); Laodelphaxstriatellus Fallen (smaller brown planthopper); Leptoglossus corculusSay (leaf-footed pine seed bug); Leptodictya tabida Herrich-Schaeffer(sugarcane lace bug); Lipaphis erysimi Kaltenbach (turnip aphid);Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolarisPalisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Westerntarnished plant bug); L. pratensis Linnaeus (common meadow bug); L.rugulipennis Poppius (European tarnished plant bug); Macrosiphumeuphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Forbes(aster leafhopper); Magicicada septendecim Linnaeus (periodical cicada);Mahanarva fimbriolata St

l (sugarcane spittlebug); M. posticata St

l (little cicada of sugarcane); Melanaphis sacchari Zehntner (sugarcaneaphid); Melanaspis glomerata Green (black scale); Metopolophium dirhodumWalker (rose grain aphid); Myzus persicae Sulzer (peach-potato aphid,green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus St

l (rice leafhopper); Nezara viridula Linnaeus (southern green stinkbug); Nilaparvata lugens St

l (brown planthopper); Nysius ericae Schilling (false chinch bug);Nysius raphanus Howard (false chinch bug); Oebalus pugnax Fabricius(rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug);Orthops campestris Linnaeus; Pemphigus spp. (root aphids and gallaphids); Peregrinus maidis Ashmead (corn planthopper); Perkinsiellasaccharicida Kirkaldy (sugarcane delphacid); Phylloxera devastatrixPergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug);Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatusFabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter(cotton fleahopper); Pseudococcus spp. (other mealybug complex);Pulvinaria elongata Newstead (cottony grass scale); Pyrilla perpusillaWalker (sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotusperniciosus Comstock (San Jose scale); Reduviidae spp.; Rhopalosiphummaidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oataphid); Saccharicoccus sacchari Cockerell (pink sugarcane mealybug);Scaptacoris castanea Perty (brown root stink bug); Schizaphis graminumRondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid);Sitobion avenae Fabricius (English grain aphid); Sogatella furciferaHorvath (white-backed planthopper); Sogatodes oryzicola Muir (ricedelphacid); Spanagonicus albofasciatus Reuter (whitemarked fleahopper);Therioaphis maculata Buckton (spotted alfalfa aphid); Tinidae spp.;Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T.citricida Kirkaldy (brown citrus aphid); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Trioza diospyri Ashmead (persimmon psylla); and Typhlocybapomaria McAtee (white apple leafhopper).

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Panonychus ulmi Koch(European red mite); Petrobia latens Muller (brown wheat mite);Steneotarsonemus bancrofti Michael (sugarcane stalk mite); spider mitesand red mites in the family Tetranychidae, Oligonychus grypus Baker &Pritchard, O. indicus Hirst (sugarcane leaf mite), O. pratensis Banks(Banks grass mite), O. stickneyi McGregor (sugarcane spider mite);Tetranychus urticae Koch (two spotted spider mite); T. mcdanieliMcGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spidermite); T. turkestani Ugarov & Nikolski (strawberry spider mite), flatmites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrusflat mite); rust and bud mites in the family Eriophyidae and otherfoliar feeding mites and mites important in human and animal health,i.e. dust mites in the family Epidermoptidae, follicle mites in thefamily Demodicidae, grain mites in the family Glycyphagidae, ticks inthe order Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclusNeumann (Australian paralysis tick); Dermacentor variabilis Say(American dog tick); Amblyomma americanum Linnaeus (lone star tick); andscab and itch mites in the families Psoroptidae, Pyemotidae, andSarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); andthe Latrodectus mactans Fabricius (black widow spider); and centipedesin the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus(house centipede). In addition, insect pests of the order Isoptera areof interest, including those of the termitidae family, such as, but notlimited to, Cornitermes cumulans Kollar, Cylindrotermes nordenskioeldiHolmgren and Pseudacanthotermes militaris Hagen (sugarcane termite); aswell as those in the Rhinotermitidae family including, but not limitedto Heterotermes tenuis Hagen. Insects of the order Thysanoptera are alsoof interest, including but not limited to thrips, such asStenchaetothrips minutus van Deventer (sugarcane thrips).

The insect pests can be adults, larvae or even ova. A preferreddevelopmental stage for testing for pesticidal activity is larvae orother immature form of the insect pest. Methods of rearing insect larvaeand performing bioassays are well known in the art. See, e.g., Czapla &Lang (1990) J. Econ. Entomol. 83:2480-2485; Griffith & Smith (1977) J.Aust. Ent. Soc. 16:366; Keiper & Foote (1996) Hydrobiologia 339:137-139;and U.S. Pat. No. 5,351,643. For example, insect pests can be reared intotal darkness at about 20° C. to about 30° C. and from about 30% toabout 70% relative humidity.

The novel chimeric pesticidal polypeptides can exhibit improvedpesticidal activity when compared to a pesticidal polypeptide lacking asolubility-enhancing polypeptide. As used herein, the term “improvedpesticidal activity” refers to a polypeptide that has enhancedpesticidal activity following the presently disclosed methods relativeto the activity of the corresponding pesticidal polypeptide lacking asolubility-enhancing polypeptide as made by the methods disclosed hereinand/or to a polypeptide that is effective against a broader range ofpests, and/or a polypeptide having specificity for a pest that is notsusceptible to the toxicity of the polypeptide prior to modification ofits sequence using the presently disclosed methods. A finding ofimproved or enhanced pesticidal activity requires a demonstration of anincrease of pesticidal activity of at least 10%, against the pest targetor at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%,200% or 300% or greater increase of pesticidal activity relative to thepesticidal activity of the polypeptide prior to the addition of asolubility-enhancing polypeptide, as determined against the same pest.

For example, an improved pesticidal activity is provided where a wideror narrower range of pests is impacted by the polypeptide relative tothe range of pests that is affected by the polypeptide prior to sequencemodification. A wider range of impact may be desirable where versatilityis desired, while a narrower range of impact may be desirable where, forexample, beneficial insects might otherwise be impacted by use orpresence of the pesticidal polypeptide. While the invention is not boundby any particular mechanism of action, an improved pesticidal activitymay also be provided by changes in one or more characteristics of apolypeptide; for example, the stability or longevity of a polypeptide inan insect gut may be increased relative to the stability or longevity ofa corresponding wild-type protein.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e. with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

As noted above, the methods involve generating novel chimeric pesticidalpolypeptide sequences and the nucleotide sequences that encode suchpolypeptides. The novel chimeric pesticidal polypeptide sequences areproduced by making a fusion protein comprising an amino acid sequence ofa solubility-enhancing polypeptide operably linked to an amino acidsequence of a pesticidal protein, particularly a cry protein.

Any pesticidal protein can be used in the presently disclosed methods.In some embodiments, the pesticidal protein is a δ-endotoxin of Bacillusspp. The specific activity of δ-endotoxins is considered highlybeneficial. Unlike most insecticides, the δ-endotoxins do not have abroad spectrum of activity, so they typically do not kill beneficialinsects. Furthermore, the δ-endotoxins are non-toxic to mammals,including humans, domesticated animals, and wildlife. In particularembodiments, the δ-endotoxin is a Cry protein.

It is well known that naturally occurring δ-endotoxins are synthesizedby B. thuringiensis sporulating cells as a proteinaceous crystallineinclusion protoxin. Upon being ingested by susceptible insect larvae,the microcrystals dissolve in the midgut, and the protoxin istransformed into a biologically active moiety by proteasescharacteristic of digestive enzymes located in the insect gut. Theactivated δ-endotoxin binds with high affinity to protein receptors onbrush-border membrane vesicles. The epithelial cells lining the midgutare the primary target of the endotoxin and are rapidly destroyed as aconsequence of membrane perforation resulting from the formation ofgated, cation-selective channels by the toxin. Both Cry and Cyt toxinsare pore-forming toxins. The α-helix regions of the Cry toxins form thetrans-membrane pore, whereas Cyt toxins insert into the membrane byforming a β-barrel comprised of β-sheets from each monomer.

Bt Cry proteins have five conserved sequence domains, and threeconserved structural domains (see, e.g., de Maagd et al. (2001) TrendsGenetics 17:193-199). The most amino-terminal conserved structuraldomain (Domain I) consists of seven alpha helices, with a centralhydrophobic helix-α5 encircled by six other amphipathic helices, and isinvolved in membrane insertion and pore formation. The second conservedstructural domain (Domain II) consists of three antiparallel beta-sheetsimplicated in cell binding, and the most carboxy-terminal conservedstructural domain (Domain III) consists of a beta-sandwich. Exposedregions in domains II and III are involved in receptor recognition andbinding, and are therefore considered determinants of toxin specificity.The location and properties of these domains are known to those of skillin the art. See, for example, Grochulski et al. (1995) J Mol Biol254:447-464; Morse, Yamamoto, and Stroud (2001) Structure 9:409-417; Liet al. (1991) Nature 353:815-821; Galitsky et al. (2001) Acta CrystD57:1101-1109; Boonserm et al. (2006) J Bacteriol 188:3391-3401;Boonserm et al. (2005) J Mol Biol 348:363-382; and Guo et al. (2009) JStruct Biol 168:259-266.

Bt Cyt proteins have a single α-β domain comprising two outer layers ofα-helix hairpins wrapped around a β-sheet (Li, Koni, and Ellar (1996) JMol Biol 257:129-152; and Cohen et al. (2008) J Mol Biol 380:820-827).The β-sheet is involved in membrane insertion.

A list of some known δ-endotoxins (Cry and Cyt endotoxins) and theirGenBank Accession Nos. are listed in Table 2, which can be used as asource for nucleic and amino acid sequences for use in the methodsdisclosed herein.

TABLE 2 Endotoxin Accession # Endotoxin Accession # Cry1Aa1 AAA22353Cry1Aa2 AAA22552 Cry1Aa3 BAA00257 Cry1Aa4 CAA31886 Cry1Aa5 BAA04468Cry1Aa6 AAA86265 Cry1Aa7 AAD46139 Cry1Aa8 I26149 Cry1Aa9 BAA77213Cry1Aa10 AAD55382 Cry1Aa11 CAA70856 Cry1Aa12 AAP80146 Cry1Aa13 AAM44305Cry1Aa14 AAP40639 Cry1Aa15 AAY66993 Cry1Aa16 HQ439776 Cry1Aa17 HQ439788Cry1Aa18 HQ439790 Cry1Aa19 HQ685121 Cry1Aa20 JF340156 Cry1Aa21 JN651496Cry1Aa22 KC158223 Cry1Ab1 AAA22330 Cry1Ab2 AAA22613 Cry1Ab3 AAA22561Cry1Ab4 BAA00071 Cry1Ab5 CAA28405 Cry1Ab6 AAA22420 Cry1Ab7 CAA31620Cry1Ab8 AAA22551 Cry1Ab9 CAA38701 Cry1Ab10 A29125 Cry1Ab11 I12419Cry1Ab12 AAC64003 Cry1Ab13 AAN76494 Cry1Ab14 AAG16877 Cry1Ab15 AAO13302Cry1Ab16 AAK55546 Cry1Ab17 AAT46415 Cry1Ab18 AAQ88259 Cry1Ab19 AAW31761Cry1Ab20 ABB72460 Cry1Ab21 ABS18384 Cry1Ab22 ABW87320 Cry1Ab23 HQ439777Cry1Ab24 HQ439778 Cry1Ab25 HQ685122 Cry1Ab26 HQ847729 Cry1Ab27 JN135249Cry1Ab28 JN135250 Cry1Ab29 JN135251 Cry1Ab30 JN135252 Cry1Ab31 JN135253Cry1Ab32 JN135254 Cry1Ab33 AAS93798 Cry1Ab34 KC156668 Cry1Ab-likeAAK14336 Cry1Ab-like AAK14337 Cry1Ab-like AAK14338 Cry1Ab-like ABG88858Cry1Ac1 AAA22331 Cry1Ac2 AAA22338 Cry1Ac3 CAA38098 Cry1Ac4 Cry1Ac4Cry1Ac5 AAA22339 Cry1Ac6 AAA86266 Cry1Ac7 AAB46989 Cry1Ac8 AAC44841Cry1Ac9 AAB49768 Cry1Ac10 CAA05505 Cry1Ac11 CAA10270 Cry1Ac12 I12418Cry1Ac13 AAD38701 Cry1Ac14 AAQ06607 Cry1Ac15 AAN07788 Cry1Ac16 AAU87037Cry1Ac17 AAX18704 Cry1Ac18 AAY88347 Cry1Ac19 ABD37053 Cry1Ac20 ABB89046Cry1Ac21 AAY66992 Cry1Ac22 ABZ01836 Cry1Ac23 CAQ30431 Cry1Ac24 ABL01535Cry1Ac25 FJ513324 Cry1Ac26 FJ617446 Cry1Ac27 FJ617447 Cry1Ac28 ACM90319Cry1Ac29 DQ438941 Cry1Ac30 GQ227507 Cry1Ac31 GU446674 Cry1Ac32 HM061081Cry1Ac33 GQ866913 Cry1Ac34 HQ230364 Cry1Ac35 JF340157 Cry1Ac36 JN387137Cry1Ac37 JQ317685 Cry1Ad1 AAA22340 Cry1Ad2 CAA01880 Cry1Ae1 AAA22410Cry1Af1 AAB82749 Cry1Ag1 AAD46137 Cry1Ah1 AAQ14326 Cry1Ah2 ABB76664Cry1Ah3 HQ439779 Cry1Ai1 AAO39719 Cry1Ai2 HQ439780 Cry1A-like AAK14339Cry1Ba1 CAA29898 Cry1Ba2 CAA65003 Cry1Ba3 AAK63251 Cry1Ba4 AAK51084Cry1Ba5 AB020894 Cry1Ba6 ABL60921 Cry1Ba7 HQ439781 Cry1Bb1 AAA22344Cry1Bb2 HQ439782 Cry1Bc1 CAA86568 Cry1Bd1 AAD10292 Cry1Bd2 AAM93496Cry1Be1 AAC32850 Cry1Be2 AAQ52387 Cry1Be3 ACV96720 Cry1Be4 HM070026Cry1Bf1 CAC50778 Cry1Bf2 AAQ52380 Cry1Bg1 AAO39720 Cry1Bh1 HQ589331Cry1Bi1 KC156700 Cry1Ca1 CAA30396 Cry1Ca2 CAA31951 Cry1Ca3 AAA22343Cry1Ca4 CAA01886 Cry1Ca5 CAA65457 Cry1Ca6 AAF37224 Cry1Ca7 AAG50438Cry1Ca8 AAM00264 Cry1Ca9 AAL79362 Cry1Ca10 AAN16462 Cry1Ca11 AAX53094Cry1Ca12 HM070027 Cry1Ca13 HQ412621 Cry1Ca14 JN651493 Cry1Cb1 M97880Cry1Cb2 AAG35409 Cry1Cb3 ACD50894 Cry1Cb-like AAX63901 Cry1Da1 CAA38099Cry1Da2 I76415 Cry1Da3 HQ439784 Cry1Db1 CAA80234 Cry1Db2 AAK48937Cry1Dc1 ABK35074 Cry1Ea1 CAA37933 Cry1Ea2 CAA39609 Cry1Ea3 AAA22345Cry1Ea4 AAD04732 Cry1Ea5 A15535 Cry1Ea6 AAL50330 Cry1Ea7 AAW72936Cry1Ea8 ABX11258 Cry1Ea9 HQ439785 Cry1Ea10 ADR00398 Cry1Ea11 JQ652456Cry1Eb1 AAA22346 Cry1Fa1 AAA22348 Cry1Fa2 AAA22347 Cry1Fa3 HM070028Cry1Fa4 HM439638 Cry1Fb1 CAA80235 Cry1Fb2 BAA25298 Cry1Fb3 AAF21767Cry1Fb4 AAC10641 Cry1Fb5 AAO13295 Cry1Fb6 ACD50892 Cry1Fb7 ACD50893Cry1Ga1 CAA80233 Cry1Ga2 CAA70506 Cry1Gb1 AAD10291 Cry1Gb2 AAO13756Cry1Gc1 AAQ52381 Cry1Ha1 CAA80236 Cry1Hb1 AAA79694 Cry1Hb2 HQ439786Cry1H-like AAF01213 Cry1Ia1 CAA44633 Cry1Ia2 AAA22354 Cry1Ia3 AAC36999Cry1Ia4 AAB00958 Cry1Ia5 CAA70124 Cry1Ia6 AAC26910 Cry1Ia7 AAM73516Cry1Ia8 AAK66742 Cry1Ia9 AAQ08616 Cry1Ia10 AAP86782 Cry1Ia11 CAC85964Cry1Ia12 AAV53390 Cry1Ia13 ABF83202 Cry1Ia14 ACG63871 Cry1Ia15 FJ617445Cry1Ia16 FJ617448 Cry1Ia17 GU989199 Cry1Ia18 ADK23801 Cry1Ia19 HQ439787Cry1Ia20 JQ228426 Cry1Ia21 JQ228424 Cry1Ia22 JQ228427 Cry1Ia23 JQ228428Cry1Ia24 JQ228429 Cry1Ia25 JQ228430 Cry1Ia26 JQ228431 Cry1Ia27 JQ228432Cry1Ia28 JQ228433 Cry1Ia29 JQ228434 Cry1Ia30 JQ317686 Cry1Ia31 JX944038Cry1Ia32 JX944039 Cry1Ia33 JX944040 Cry1Ib1 AAA82114 Cry1Ib2 ABW88019Cry1Ib3 ACD75515 Cry1Ib4 HM051227 Cry1Ib5 HM070028 Cry1Ib6 ADK38579Cry1Ib7 JN571740 Cry1Ib8 JN675714 Cry1Ib9 JN675715 Cry1Ib10 JN675716Cry1Ib11 JQ228423 Cry1Ic1 AAC62933 Cry1Ic2 AAE71691 Cry1Id1 AAD44366Cry1Id2 JQ228422 Cry1Ie1 AAG43526 Cry1Ie2 HM439636 Cry1Ie3 KC156647Cry1Ie4 KC156681 Cry1If1 AAQ52382 Cry1Ig1 KC156701 Cry1I-like AAC31094Cry1I-like ABG88859 Cry1Ja1 AAA22341 Cry1Ja2 HM070030 Cry1Ja3 JQ228425Cry1Jb1 AAA98959 Cry1Jc1 AAC31092 Cry1Jc2 AAQ52372 Cry1Jd1 CAC50779Cry1Ka1 AAB00376 Cry1Ka2 HQ439783 Cry1La1 AAS60191 Cry1La2 HM070031Cry1Ma1 FJ884067 Cry1Ma2 KC156659 Cry1Na1 KC156648 Cry1Nb1 KC156678Cry1-like AAC31091 Cry2Aa1 AAA22335 Cry2Aa2 AAA83516 Cry2Aa3 D86064Cry2Aa4 AAC04867 Cry2Aa5 CAA10671 Cry2Aa6 CAA10672 Cry2Aa7 CAA10670Cry2Aa8 AA013734 Cry2Aa9 AAO13750 Cry2Aa10 AAQ04263 Cry2Aa11 AAQ52384Cry2Aa12 ABI83671 Cry2Aa13 ABL01536 Cry2Aa14 ACF04939 Cry2Aa15 JN426947Cry2Ab1 AAA22342 Cry2Ab2 CAA39075 Cry2Ab3 AAG36762 Cry2Ab4 AAO13296Cry2Ab5 AAQ04609 Cry2Ab6 AAP59457 Cry2Ab7 AAZ66347 Cry2Ab8 ABC95996Cry2Ab9 ABC74968 Cry2Ab10 EF157306 Cry2Ab11 CAM84575 Cry2Ab12 ABM21764Cry2Ab13 ACG76120 Cry2Ab14 ACG76121 Cry2Ab15 HM037126 Cry2Ab16 GQ866914Cry2Ab17 HQ439789 Cry2Ab18 JN135255 Cry2Ab19 JN135256 Cry2Ab20 JN135257Cry2Ab21 JN135258 Cry2Ab22 JN135259 Cry2Ab23 JN135260 Cry2Ab24 JN135261Cry2Ab25 JN415485 Cry2Ab26 JN426946 Cry2Ab27 JN415764 Cry2Ab28 JN651494Cry2Ac1 CAA40536 Cry2Ac2 AAG35410 Cry2Ac3 AAQ52385 Cry2Ac4 ABC95997Cry2Ac5 ABC74969 Cry2Ac6 ABC74793 Cry2Ac7 CAL18690 Cry2Ac8 CAM09325Cry2Ac9 CAM09326 Cry2Ac10 ABN15104 Cry2Ac11 CAM83895 Cry2Ac12 CAM83896Cry2Ad1 AAF09583 Cry2Ad2 ABC86927 Cry2Ad3 CAK29504 Cry2Ad4 CAM32331Cry2Ad5 CAO78739 Cry2Ae1 AAQ52362 Cry2Af1 ABO30519 Cry2Af2 GQ866915Cry2Ag1 ACH91610 Cry2Ah1 EU939453 Cry2Ah2 ACL80665 Cry2Ah3 GU073380Cry2Ah4 KC156702 Cry2Ai1 FJ788388 Cry2Aj Cry2Ak1 KC156660 Cry2Ba1KC156658 Cry3Aa1 AAA22336 Cry3Aa2 AAA22541 Cry3Aa3 CAA68482 Cry3Aa4AAA22542 Cry3Aa5 AAA50255 Cry3Aa6 AAC43266 Cry3Aa7 CAB41411 Cry3Aa8AAS79487 Cry3Aa9 AAW05659 Cry3Aa10 AAU29411 Cry3Aa11 AAW82872 Cry3Aa12ABY49136 Cry3Ba1 CAA34983 Cry3Ba2 CAA00645 Cry3Ba3 JQ397327 Cry3Bb1AAA22334 Cry3Bb2 AAA74198 Cry3Bb3 I15475 Cry3Ca1 CAA42469 Cry4Aa1CAA68485 Cry4Aa2 BAA00179 Cry4Aa3 CAD30148 Cry4Aa4 AFB18317 Cry4A-likeAAY96321 Cry4Ba1 CAA30312 Cry4Ba2 CAA30114 Cry4Ba3 AAA22337 Cry4Ba4BAA00178 Cry4Ba5 CAD30095 Cry4Ba-like ABC47686 Cry4Ca1 EU646202 Cry4Cb1FJ403208 Cry4Cb2 FJ597622 Cry4Cc1 FJ403207 Cry5Aa1 AAA67694 Cry5Ab1AAA67693 Cry5Ac1 I34543 Cry5Ad1 ABQ82087 Cry5Ba1 AAA68598 Cry5Ba2ABW88931 Cry5Ba3 AFJ04417 Cry5Ca1 HM461869 Cry5Ca2 ZP_04123426 Cry5Da1HM461870 Cry5Da2 ZP_04123980 Cry5Ea1 HM485580 Cry5Ea2 ZP_04124038Cry6Aa1 AAA22357 Cry6Aa2 AAM46849 Cry6Aa3 ABH03377 Cry6Ba1 AAA22358Cry7Aa1 AAA22351 Cry7Ab1 AAA21120 Cry7Ab2 AAA21121 Cry7Ab3 ABX24522Cry7Ab4 EU380678 Cry7Ab5 ABX79555 Cry7Ab6 AC144005 Cry7Ab7 ADB89216Cry7Ab8 GU145299 Cry7Ab9 ADD92572 Cry7Ba1 ABB70817 Cry7Bb1 KC156653Cry7Ca1 ABR67863 Cry7Cb1 KC156698 Cry7Da1 ACQ99547 Cry7Da2 HM572236Cry7Da3 KC156679 Cry7Ea1 HM035086 Cry7Ea2 HM132124 Cry7Ea3 EEM19403Cry7Fa1 HM035088 Cry7Fa2 EEM19090 Cry7Fb1 HM572235 Cry7Fb2 KC156682Cry7Ga1 HM572237 Cry7Ga2 KC156669 Cry7Gb1 KC156650 Cry7Gc1 KC156654Cry7Gd1 KC156697 Cry7Ha1 KC156651 Cry71a1 KC156665 Cry7Ja1 KC156671Cry7Ka1 KC156680 Cry7Kb1 BAM99306 Cry7La1 BAM99307 Cry8Aa1 AAA21117Cry8Ab1 EU044830 Cry8Ac1 KC156662 Cry8Ad1 KC156684 Cry8Ba1 AAA21118Cry8Bb1 CAD57542 Cry8Bc1 CAD57543 Cry8Ca1 AAA21119 Cry8Ca2 AAR98783Cry8Ca3 EU625349 Cry8Ca4 ADB54826 Cry8Da1 BAC07226 Cry8Da2 BD133574Cry8Da3 BD133575 Cry8Db1 BAF93483 Cry8Ea1 AAQ73470 Cry8Ea2 EU047597Cry8Ea3 KC855216 Cry8Fa1 AAT48690 Cry8Fa2 HQ174208 Cry8Fa3 AFH78109Cry8Ga1 AAT46073 Cry8Ga2 ABC42043 Cry8Ga3 FJ198072 Cry8Ha1 AAW81032Cry81a1 EU381044 Cry81a2 GU073381 Cry81a3 HM044664 Cry81a4 KC156674Cry81b1 GU325772 Cry81b2 KC156677 Cry8Ja1 EU625348 Cry8Ka1 FJ422558Cry8Ka2 ACN87262 Cry8Kb1 HM123758 Cry8Kb2 KC156675 Cry8La1 GU325771Cry8Ma1 HM044665 Cry8Ma2 EEM86551 Cry8Ma3 HM210574 Cry8Na1 HM640939Cry8Pa1 HQ388415 Cry8Qa1 HQ441166 Cry8Qa2 KC152468 Cry8Ra1 AFP87548Cry8Sa1 JQ740599 Cry8Ta1 KC156673 Cry8-like FJ770571 Cry8-like ABS53003Cry9Aa1 CAA41122 Cry9Aa2 CAA41425 Cry9Aa3 GQ249293 Cry9Aa4 GQ249294Cry9Aa5 JX174110 Cry9Aa like AAQ52376 Cry9Ba1 CAA52927 Cry9Ba2 GU299522Cry9Bb1 AAV28716 Cry9Ca1 CAA85764 Cry9Ca2 AAQ52375 Cry9Da1 BAA19948Cry9Da2 AAB97923 Cry9Da3 GQ249293 Cry9Da4 GQ249297 Cry9Db1 AAX78439Cry9Dc1 KC156683 Cry9Ea1 BAA34908 Cry9Ea2 AAO12908 Cry9Ea3 ABM21765Cry9Ea4 ACE88267 Cry9Ea5 ACF04743 Cry9Ea6 ACG63872 Cry9Ea7 FJ380927Cry9Ea8 GQ249292 Cry9Ea9 JN651495 Cry9Eb1 CAC50780 Cry9Eb2 GQ249298Cry9Eb3 KC156646 Cry9Ec1 AAC63366 Cry9Ed1 AAX78440 Cry9Ee1 GQ249296Cry9Ee2 KC156664 Cry9Fa1 KC156692 Cry9Ga1 KC156699 Cry9-like AAC63366Cry10Aa1 AAA22614 Cry10Aa2 E00614 Cry10Aa3 CAD30098 Cry10Aa4 AFB18318Cry10A-like DQ167578 Cry11Aa1 AAA22352 Cry11Aa2 AAA22611 Cry11Aa3CAD30081 Cry11Aa4 AFB18319 Cry11Aa-like DQ166531 Cry11Ba1 CAA60504Cry11Bb1 AAC97162 Cry11Bb2 HM068615 Cry12Aa1 AAA22355 Cry13Aa1 AAA22356Cry14Aa1 AAA21516 Cry14Ab1 KC156652 Cry15Aa1 AAA22333 Cry16Aa1 CAA63860Cry17Aa1 CAA67841 Cry18Aa1 CAA67506 Cry18Ba1 AAF89667 Cry18Ca1 AAF89668Cry19Aa1 CAA68875 Cry19Ba1 BAA32397 Cry19Ca1 AFM37572 Cry20Aa1 AAB93476Cry20Ba1 ACS93601 Cry20Ba2 KC156694 Cry20-like GQ144333 Cry21Aa1 I32932Cry21Aa2 I66477 Cry21Ba1 BAC06484 Cry21Ca1 JF521577 Cry21Ca2 KC156687Cry21Da1 JF521578 Cry22Aa1 I34547 Cry22Aa2 CAD43579 Cry22Aa3 ACD93211Cry22Ab1 AAK50456 Cry22Ab2 CAD43577 Cry22Ba1 CAD43578 Cry22Bb1 KC156672Cry23Aa1 AAF76375 Cry24Aa1 AAC61891 Cry24Ba1 BAD32657 Cry24Ca1 CAJ43600Cry25Aa1 AAC61892 Cry26Aa1 AAD25075 Cry27Aa1 BAA82796 Cry28Aa1 AAD24189Cry28Aa2 AAG00235 Cry29Aa1 CAC80985 Cry30Aa1 CAC80986 Cry30Ba1 BAD00052Cry30Ca1 BAD67157 Cry30Ca2 ACU24781 Cry30Da1 EF095955 Cry30Db1 BAE80088Cry30Ea1 ACC95445 Cry30Ea2 FJ499389 Cry30Fa1 ACI22625 Cry30Ga1 ACG60020Cry30Ga2 HQ638217 Cry31Aa1 BAB11757 Cry31Aa2 AAL87458 Cry31Aa3 BAE79808Cry31Aa4 BAF32571 Cry31Aa5 BAF32572 Cry31Aa6 BAI44026 Cry31Ab1 BAE79809Cry31Ab2 BAF32570 Cry31Ac1 BAF34368 Cry31Ac2 AB731600 Cry31Ad1 BAI44022Cry32Aa1 AAG36711 Cry32Aa2 GU063849 Cry32Ab1 GU063850 Cry32Ba1 BAB78601Cry32Ca1 BAB78602 Cry32Cb1 KC156708 Cry32Da1 BAB78603 Cry32Ea1 GU324274Cry32Ea2 KC156686 Cry32Eb1 KC156663 Cry32Fa1 KC156656 Cry32Ga1 KC156657Cry32Ha1 KC156661 Cry32Hb1 KC156666 Cry32Ia1 KC156667 Cry32Ja1 KC156685Cry32Ka1 KC156688 Cry32La1 KC156689 Cry32Ma1 KC156690 Cry32Mb1 KC156704Cry32Na1 KC156691 Cry32Oa1 KC156703 Cry32Pa1 KC156705 Cry32Qa1 KC156706Cry32Ra1 KC156707 Cry32Sa1 KC156709 Cry32Ta1 KC156710 Cry32Ua1 KC156655Cry33Aa1 AAL26871 Cry34Aa1 AAG50341 Cry34Aa2 AAK64560 Cry34Aa3 AAT29032Cry34Aa4 AAT29030 Cry34Ab1 AAG41671 Cry34Ac1 AAG50118 Cry34Ac2 AAK64562Cry34Ac3 AAT29029 Cry34Ba1 AAK64565 Cry34Ba2 AAT29033 Cry34Ba3 AAT29031Cry35Aa1 AAG50342 Cry35Aa2 AAK64561 Cry35Aa3 AAT29028 Cry35Aa4 AAT29025Cry35Ab1 AAG41672 Cry35Ab2 AAK64563 Cry35Ab3 AY536891 Cry35Ac1 AAG50117Cry35Ba1 AAK64566 Cry35Ba2 AAT29027 Cry35Ba3 AAT29026 Cry36Aa1 AAK64558Cry37Aa1 AAF76376 Cry38Aa1 AAK64559 Cry39Aa1 BAB72016 Cry40Aa1 BAB72018Cry40Ba1 BAC77648 Cry40Ca1 EU381045 Cry40Da1 ACF15199 Cry41Aa1 BAD35157Cry41Ab1 BAD35163 Cry41Ba1 HM461871 Cry41Ba2 ZP_04099652 Cry42Aa1BAD35166 Cry43Aa1 BAD15301 Cry43Aa2 BAD95474 Cry43Ba1 BAD15303 Cry43Ca1KC156676 Cry43Cb1 KC156695 Cry43Cc1 KC156696 Cry43-like BAD15305 Cry44AaCry44Aa Cry45Aa BAD22577 Cry46Aa BAC79010 Cry46Aa2 BAG68906 Cry46AbBAD35170 Cry47Aa AAY24695 Cry48Aa CAJ18351 Cry48Aa2 CAJ86545 Cry48Aa3CAJ86546 Cry48Ab CAJ86548 Cry48Ab2 CAJ86549 Cry49Aa CAH56541 Cry49Aa2CAJ86541 Cry49Aa3 CAJ86543 Cry49Aa4 CAJ86544 Cry49Ab1 CAJ86542 Cry50Aa1BAE86999 Cry50Ba1 GU446675 Cry50Ba2 GU446676 Cry51Aa1 ABI14444 Cry51Aa2GU570697 Cry52Aa1 EF613489 Cry52Ba1 FJ361760 Cry53Aa1 EF633476 Cry53Ab1FJ361759 Cry54Aa1 ACA52194 Cry54Aa2 GQ140349 Cry54Ba1 GU446677 Cry55Aa1ABW88932 Cry54Ab1 JQ916908 Cry55Aa2 AAE33526 Cry56Aa1 ACU57499 Cry56Aa2GQ483512 Cry56Aa3 JX025567 Cry57Aa1 ANC87261 Cry58Aa1 ANC87260 Cry59Ba1JN790647 Cry59Aa1 ACR43758 Cry60Aa1 ACU24782 Cry60Aa2 EAO57254 Cry60Aa3EEM99278 Cry60Ba1 GU810818 Cry60Ba2 EAO57253 Cry60Ba3 EEM99279 Cry61Aa1HM035087 Cry61Aa2 HM132125 Cry61Aa3 EEM19308 Cry62Aa1 HM054509 Cry63Aa1BAI44028 Cry64Aa1 BAJ05397 Cry65Aa1 HM461868 Cry65Aa2 ZP_04123838Cry66Aa1 HM485581 Cry66Aa2 ZP_04099945 Cry67Aa1 HM485582 Cry67Aa2ZP_04148882 Cry68Aa1 HQ113114 Cry69Aa1 HQ401006 Cry69Aa2 JQ821388Cry69Ab1 JN209957 Cry70Aa1 JN646781 Cry70Ba1 ADO51070 Cry70Bb1 EEL67276Cry71Aa1 JX025568 Cry72Aa1 JX025569 Cyt1Aa X03182 Cyt1Ab X98793 Cyt1BU37196 Cyt2A Z14147 Cyt2B U52043 * The amino acid and correspondingnucleotide sequences of the accession numbers in Table 2 are hereinincorporated by reference.

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of cryproteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318;Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No.7,064,249); a Cry3A protein including but not limited to an engineeredhybrid insecticidal protein (eHIP) created by fusing unique combinationsof variable regions and conserved blocks of at least two different Cryproteins (US Patent Application Publication Number 2010/0017914); a Cry4protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos.7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B,Cry9C, Cry9D, Cry9E and Cry9F families; a Cry15 protein of Naimov, etal., (2008) Applied and Environmental Microbiology, 74:7145-7151; aCry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535,6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 andCryET34 homologs of US Patent Publication Number 2006/0191034,2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US Patent Application Publication Number 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494, AXMI-027,AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039,AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 andAXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO2005/021585; AXMI-008 of US Patent Application Publication Number2004/0250311; AXMI-006 of US Patent Application Publication Number2004/0216186; AXMI-007 of US Patent Application Publication Number2004/0210965; AXMI-009 of US Patent Application Number 2004/0210964;AXMI-014 of US Patent Application Publication Number 2004/0197917;AXMI-004 of US Patent Application Publication Number 2004/0197916;AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008,AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462;AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US Patent ApplicationPublication Number 2011/0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015,AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022,AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent ApplicationPublication Number 2011/0263488; AXMI-R1 and related proteins of USPatent Application Publication Number 2010/0197592; AXMI221Z, AXMI222z,AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219,AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S.Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045of US Patent Application Publication Number 2010/0298211; AXMI-066 andAXMI-076 of US Patent Application Publication Number 2009/0144852;AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143,AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155,AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168,AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176,AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186,AXMI187, AXM1188, AXM1189 of U.S. Pat. No. 8,318,900; AXM1079, AXM1080,AXM1081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099,AXMI100, AXMI101, AXM1102, AXM1103, AXM1104, AXM1107, AXM1108, AXM1109,AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119,AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268,AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138,AXMI137 of US Patent Application Publication Number 2010/0005543, cryproteins such as Cry1A and Cry3A having modified proteolytic sites ofU.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein fromBacillus thuringiensis strain VBTS 2528 of US Patent ApplicationPublication Number 2011/0064710. Other Cry proteins are well known toone skilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on theworld-wide web using the “www” prefix). The insecticidal activity of Cryproteins is well known to one skilled in the art (for review, see, vanFrannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to plantsexpressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2,Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A,mCry3A, Cry9c and CBI-Bt have received regulatory approval (see,Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GMCrop Database Center for Environmental Risk Assessment (CERA), ILSIResearch Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database, which can be accessed onthe world-wide web using the “www” prefix).

The primary structure of a pesticidal polypeptide can be used in itsnative form or modified in order to introduce amino acid residues(either through addition or substitution) that contribute to or aresuspected of contributing to the pesticidal activity of at least onepesticidal polypeptide. Functional data and results fromstructure-function analyses can be consulted to identify sequencesand/or amino acid residues that contribute to pesticidal activity thatshould be retained or introduced into the candidate polypeptidesequence. Those residues that contribute to or are suspected ofcontributing to pesticidal activity include those residues that enhanceefficacy or those that dictate pesticidal specificity, including thoseresidues that narrow or broaden the range of pests of pesticidalproteins.

For example, the aromaticity of the tyrosine and phenylalanine residuesat position 249 and 264, respectively, in helix 7 of the Bt Cry4Ba toxinhas been found to be important for toxicity (Tiewsiri andAngsuthanasombat (2007) J Biochem Mol Biol 40:163-171). Thus, in thoseembodiments wherein the candidate polypeptide is homologous with the BtCry4Ba toxin, the residues at the positions corresponding to positions249 and 264 of Cry4Ba should be maintained if they are aromatic ormodified if they are non-aromatic.

Further, additional mutations can be introduced into pesticidalpolypeptide sequence to improve the pesticidal activity. For example, asdescribed in U.S. Pat. Nos. 7,462,760 and 7,105,332 (each of which areherein incorporated by reference in its entirety), mutations can beintroduced into the candidate polypeptide sequence to destroyproteolytic sites to protect the polypeptide from degradative digestion,for example, by plant proteases. As a further example, the toxicity ofBt Cry proteins can be improved by introducing at least one moreprotease-sensitive site (e.g., trypsin cleavage site) into the regionlocated between alpha helices 3 and 4 of domain 1 of the endotoxinprotein (see U.S. Patent Application Publication No. US2004/0091505,which is herein incorporated by reference in its entirety). As anothernon-limiting example, a protease-sensitive site that is readily cleavedby insect chymotrypsin, e.g., a chymotrypsin found in the berthaarmyworm or the corn earworm (Hegedus et al. (2003) Arch. InsectBiochem. Physiol. 53: 30-47; and Lenz et al. (1991) Arch. InsectBiochem. Physiol. 16: 201-212), may be added to the candidatepolypeptide sequence to provide improved toxicity to the polypeptide.

In certain embodiments of the invention, the Cry protein comprises theamino acid sequence set forth in SEQ ID NO: 8, 10, 12 or 14 or fragmentor variant thereof. Any nucleotide sequence encoding the amino acidsequence set forth in 8, 10, 12 or 14 or fragment or variant thereof,can be used in the methods and compositions of the present inventionincluding, but not limited to the nucleotides sequences encoding SEQ IDNOS: 8, 10, 12, and 14 which are set forth in SEQ ID NOS: 7, 9, 11, and13, respectively. In some embodiments of the invention, the nucleotidesequences of the invention will be optimized for expression in a hostorganism or cell of interest, particularly a plant, more particularly acrop plant, most particularly a maize plant.

Methods of measuring pesticidal activity by insect bioassays are wellknown in the art. See, e.g., Brooke et al. (2001) Bull. Entomol. Res.91:265-272; Chen et al. (2007) Proc. Natl. Acad. Sci. USA104:13901-13906; Crespo et al. (2008) Appl. Environ. Microb. 74:130-135;Kham bay et al. (2003) Pest Manag. Sci. 59:174-182; Liu & Dean (2006)Protein Eng. Des. Sel. 19:107-111; Marrone et al. (1985) J. Econ.Entomol. 78:290-293; Robertson et al., Pesticide Bioassays withArthropods (2^(nd) ed., CRC Press 2007); Scott & McKibben (1976) J.Econ. Entomol. 71:343-344; Strickman (1985) Bull. Environ. Contam.Toxicol. 35:133-142; and Verma et al. (1982) Water Res. 16 525-529; aswell as U.S. Pat. No. 6,268,181. Examples of insect bioassays include,but are not limited to, pest mortality, pest weight loss, pestrepellency, pest attraction, and other behavioral and physical changesof the pest after feeding and exposure to a pesticide or pesticidalpolypeptide for an appropriate length of time. General methods includeaddition of the pesticide, pesticidal polypeptide or an organism havingthe pesticidal polypeptide to the diet source in an enclosed container.See, e.g., U.S. Pat. Nos. 6,339,144 and 6,570,005.

Further, a nucleic acid encoding the novel chimeric pesticidalpolypeptide can be derived from the amino acid sequence and can begenerated using any method known in the art. Therefore, novel isolatedpesticidal polypeptides and isolated nucleic acid molecules encoding thesame are provided. An “isolated” or “purified” polynucleotide or proteinor biologically active fragment thereof, is substantially or essentiallyfree from components that normally accompany or interact with thepolynucleotide or protein as found in its naturally occurringenvironment. Thus, an isolated or purified polynucleotide or protein issubstantially free of other cellular material or culture medium whenproduced by recombinant techniques or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Optimally, an“isolated” polynucleotide is free of sequences (optimally proteinencoding sequences) that naturally flank the polynucleotide (i.e.,sequences located at the 5′ and 3′ ends of the polynucleotide) in thegenomic DNA of the organism from which the polynucleotide is derived.For example, in various embodiments, the isolated polynucleotide cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleotide sequence that naturally flank the polynucleotide ingenomic DNA of the cell from which the polynucleotide is derived. Aprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5% or 1%(by dry weight) of contaminating protein. When the protein of theinvention or biologically active fragment thereof is recombinantlyproduced, optimally culture medium represents less than about 30%, 20%,10%, 5% or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Further provided are novel chimeric pesticidal polypeptides comprisingfragments and variants of a solubility-enhancing polypeptide and/orpesticidal protein as well as nucleic acid molecules encoding such novelchimeric pesticidal polypeptides. Fragments and variants of the novelchimeric pesticidal polypeptides and chimeric pesticidalpolypeptide-encoding nucleic acid molecules are also provided. By“fragment” is intended a portion of the polynucleotide or a portion ofthe amino acid sequence and hence protein encoded thereby. Fragments ofa polynucleotide may encode protein fragments that retainsolubility-enhancing activity and/or pesticidal activity. Alternatively,fragments of a polynucleotide that are useful as hybridization probesgenerally do not encode fragment proteins retaining asolubility-enhancing and/or pesticidal activity. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the full-lengthpolynucleotide encoding the novel chimeric pesticidal polypeptides.

A fragment of a pesticide-encoding polynucleotide that encodes abiologically active fragment of a solubility-enhancing polypeptide orpesticidal protein will encode at least 15, 25, 30, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000 or 1,100 contiguous aminoacids or up to the total number of amino acids present in a chimericpesticidal polypeptide, a solubility-enhancing polypeptide or apesticidal protein of the invention. Fragments of a chimeric pesticidalpolypeptide, solubility-enhancing polypeptide or a pesticidal proteinthat are useful as hybridization probes or PCR primers generally neednot encode a biologically active fragment of a chimeric pesticidalpolypeptide, a solubility-enhancing polypeptide or a pesticidal protein.

Thus, a fragment of a chimeric pesticidal polypeptide, asolubility-enhancing polypeptide or a pesticidal protein may encode abiologically active fragment of a pesticidal protein or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods well known in the art and disclosed elsewhere herein. Abiologically active fragment of a pesticidal protein can be prepared byisolating a portion of one of the pesticide-encoding polynucleotides,expressing the encoded portion of the pesticidal protein (e.g., byrecombinant expression in vitro), and assessing the activity of theencoded portion of the pesticidal protein. Polynucleotides that arefragments of a chimeric pesticidal polypeptide, a solubility-enhancingpolypeptide or a pesticidal protein sequence comprise at least 16, 20,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2500 or 3000 contiguous nucleotides or up to the number ofnucleotides present in a full-length pesticide-encoding polynucleotidediscovered using the methods disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe chimeric pesticidal polypeptide, solubility-enhancing polypeptidesor pesticidal proteins of the invention. Naturally occurring allelicvariants such as these can be identified with the use of well-knownmolecular biology techniques, as, for example, with polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantpolynucleotides also include synthetically derived polynucleotides, suchas those generated, for example, by using site-directed mutagenesis butwhich still encode a chimeric pesticidal polypeptide, asolubility-enhancing polypeptide or a pesticidal protein of theinvention. Generally, variants of a particular polynucleotide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters as described elsewhereherein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 2 is disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion (so-called truncation) of one or more amino acids atthe N-terminal and/or C-terminal end of the native protein; deletionand/or addition of one or more amino acids at one or more internal sitesin the native protein; or substitution of one or more amino acids at oneor more sites in the native protein. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,pesticidal activity as described herein. Such variants may result from,for example, genetic polymorphism or from human manipulation.Biologically active variants of a chimeric pesticidal polypeptide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a protein of theinvention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2or even 1 amino acid residue.

The novel chimeric pesticidal polypeptide made using the presentlydisclosed methods may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions to thesolubility-enhancing polypeptide and/or the pesticidal polypeptideportion of the chimeric pesticidal polypeptide. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants and fragments of the chimeric pesticidal polypeptides,pesticidal polypeptides, and solubility-enhancing polypeptide s can beprepared by mutations in the DNA. Methods for mutagenesis andpolynucleotide alterations are well known in the art. See, for example,Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al.(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be optimal.

The deletions, insertions, and substitutions of the novel chimericpesticidal polypeptide sequences, pesticidal polypeptide sequences andsolubility-enhancing polypeptide sequences encompassed herein are notexpected to produce radical changes in the characteristics of theprotein. However, when it is difficult to predict the exact effect ofthe substitution, deletion or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. That is, the pesticidal activity can beevaluated using an insect feeding bioassays as described elsewhereherein. Preferably, the biologically activity of a solubility-enhancingpolypeptide or fragment or variant thereof is evaluated by operablylinking the solubility-enhancing polypeptide, fragment or variant to apesticidal polypeptide and assaying the pesticidal activity of theresulting chimeric pesticidal polypeptide using the methods disclosedherein or otherwise known in the art. The pesticidal activity of thechimeric pesticidal polypeptide can be compared to the pesticidalactivity of the pesticidal peptide (i.e., without thesolubility-enhancing polypeptide, fragment or variant) to determine ifthe solubility-enhancing polypeptide, fragment or variant increases theinsecticidal activity the chimeric pesticidal polypeptide. Preferably,in any insect feeding assays in which the chimeric pesticidalpolypeptide its corresponding pesticidal polypeptide, the amount of therespective polypeptides that are fed to the insects will be adjusted totake into the account the large molecular weight of the chimericpesticidal polypeptide such that pesticidal activity comparisons willeffectively be made on a per molecule or per mole basis. Furthermore, itis recognized that insecticidal activity of chimeric pesticidalpolypeptides with different solubility-enhancing polypeptides orfragments or variants of a solubility-enhancing polypeptide can alsoevaluated and compared to one another in a like manner and preferablywith adjustments for any differences in molecular weights between thechimeric pesticidal polypeptides that are being compared.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more differentpesticide-coding sequences can be manipulated to create a new pesticidalpolypeptide possessing the desired properties and these new pesticidalproteins can be used in the methods disclosed herein to make chimericpesticidal polypepties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides, and may be labeledwith a detectable group such as ³²P or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the babyboom polynucleotide. Methodsfor preparation of probes for hybridization and for construction of cDNAand genomic libraries are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, the entire pesticide-encoding polynucleotide or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding pesticide-encoding polynucleotides andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique amongpesticide-encoding polynucleotide sequences and are optimally at leastabout 10 nucleotides in length, and most optimally at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticide-encoding polynucleotides from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1× SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

Nucleic acids encoding the novel chimeric pesticidal polypeptides can beused in DNA constructs for expression in various hosts, includingplants. Thus, expression cassettes comprising the novel chimericpesticidal polypeptide-encoding nucleic acids for expression in theorganism of interest are provided. The cassette will include 5′ and 3′regulatory sequences operably linked to a novel chimeric pesticidalpolypeptide-encoding polynucleotide. “Operably linked” is intended tomean a functional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. In the case of fusionproteins or fusion polypeptides, an operable linkage between, forexample, a first amino amino sequence of interest and a second aminoacid of interest results in a single amino acid sequence, whichcomprises both the first and second amino acid sequences, and whereinthe C-terminal amino acid the first amino amino sequence is covalentlyattached to the N-terminal amino acid of the second amino acid sequenceby a peptide bond. Operably linked elements may be contiguous ornon-contiguous. The cassette may additionally contain at least oneadditional polynucleotide of interest to be cotransformed into theorganism. Alternatively, the additional polynucleotide(s) of interestcan be provided on multiple expression cassettes. Such an expressioncassette is provided with a plurality of restriction sites and/orrecombination sites for insertion of the pesticide-encodingpolynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The chimeric pesticidal polypeptides of the present invention comprisean amino acid sequence of a solubility-enhancing polypeptide operablylinked to an amino acid sequence of a Cry endotoxin. It is recognizedthat in such an operable linkage, the amino acid sequence of thesolubility-enhancing polypeptide can be contiguous to the amino acidsequence of the Cry endotoxin or can be separated by an interveninglinker or other amino acid sequence. Likewise, the chimeric pesticidalpolypeptide-encoding nucleic acid molecules of the present inventioncomprise a nucleotide sequence encoding a solubility-enhancingpolypeptide operably linked to a nucleotide sequence encoding a Cryendotoxin. It is further recognized that in such an operable linkage,the nucleotide sequence encoding the solubility-enhancing polypeptidecan be contiguous to the nucleotide sequence encoding the Cry endotoxinor can be separated by an intervening linker-encoding nucleotidesequence or other nucleotide sequence.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a novel pesticide-encoding polynucleotide, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism to which the expression cassette isintroduced. The regulatory regions (i.e., promoters, transcriptionalregulatory regions, and translational termination regions) and/or thepesticide-encoding polynucleotide may be native/analogous to the hostcell or to each other. Alternatively, the regulatory regions and/or thepesticide-encoding polynucleotide may be heterologous to the host cellor to each other. As used herein, “heterologous” in reference to asequence is a sequence that originates from a foreign species or, iffrom the same species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus orthe promoter is not the native promoter for the operably linkedpolynucleotide.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked pesticidal polynucleotideof interest, may be native with the plant host or may be derived fromanother source (i.e., foreign or heterologous) to the promoter, thepesticidal polynucleotide of interest, the plant host or any combinationthereof. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also Guerineau et al. (1991) Mol. Gen.Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al.(1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272;Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic AcidsRes. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa etal. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The chimeric pesticidal polypeptide-encoding nucleic acid molecules,expression cassettes comprising the same or the chimeric pesticidalpolypeptides can be introduced into an organism or host cell,particularly a non-human host cell. Host cells may be prokaryotic cellssuch as E. coli or eukaryotic cells such as yeast, insect, amphibian ormammalian cells. In some examples, host cells are monocotyledonous ordicotyledonous plant cells.

The nucleic acid encoding the novel chimeric pesticidal polypeptides canbe introduced into microorganisms that multiply on plants (epiphytes) todeliver chimeric pesticidal polypeptides to potential target pests.Alternatively, the chimeric pesticidal polypeptides can be directlyintroduced into such microorganisms. Epiphytes, for example, can begram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Nucleic acids encoding the pesticidal proteins of theinvention or the chimeric pesticidal polypeptides can be introduced intoa root-colonizing Bacillus cereus by standard methods known in the art.

Nucleic acids encoding chimeric pesticidal polypeptides can beintroduced, for example, into the root-colonizing Bacillus by means ofelectrotransformation. Specifically, nucleic acids encoding thepesticidal proteins can be cloned into a shuttle vector, for example,pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. Theshuttle vector pHT3101 containing the coding sequence for the particularchimeric pesticidal polypeptide-encoding nucleic acid can, for example,be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218).

Expression systems can be designed so that chimeric pesticidalpolypeptides are secreted outside the cytoplasm of gram-negativebacteria, such as E. coli, for example. Advantages of having chimericpesticidal polypeptides secreted are: (1) avoidance of potentialcytotoxic effects of the chimeric pesticidal polypeptides expressed; and(2) improvement in the efficiency of purification of the chimericpesticidal polypeptides, including, but not limited to, increasedefficiency in the recovery and purification of the protein per volumecell broth and decreased time and/or costs of recovery and purificationper unit protein.

Chimeric pesticidal polypeptides can be made to be secreted in E. coli,for example, by fusing an appropriate E. coli signal peptide to theamino-terminal end of the pesticidal protein. Signal peptides recognizedby E. coli can be found in proteins already known to be secreted in E.coli, for example the OmpA protein (Ghrayeb et al. (1984) EMBO J,3:2437-2442). OmpA is a major protein of the E. coli outer membrane, andthus its signal peptide is thought to be efficient in the translocationprocess. Also, the OmpA signal peptide does not need to be modifiedbefore processing as may be the case for other signal peptides, forexample lipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol.153: 492).

Chimeric pesticidal polypeptides of the invention can be fermented in abacterial host and the resulting bacteria processed and used as amicrobial spray in the same manner that Bacillus thuringiensis strainshave been used as insecticidal sprays. In the case of a chimericpesticidal polypeptide(s) that is secreted from Bacillus, the secretionsignal is removed or mutated using procedures known in the art. Suchmutations and/or deletions prevent secretion of the chimeric pesticidalpolypeptide(s) into the growth medium during the fermentation process.The chimeric pesticidal polypeptides are retained within the cell, andthe cells are then processed to yield the encapsulated chimericpesticidal polypeptides. Any suitable microorganism can be used for thispurpose. Pseudomonas has been used to express Bacillus thuringiensisendotoxins as encapsulated proteins and the resulting cells processedand sprayed as an insecticide (Gaertner et al. (1993), in: AdvancedEngineered Pesticides, ed. Kim).

Alternatively, the chimeric pesticidal polypeptides are produced byintroducing a heterologous nucleic acid encoding the chimeric pesticidalpolypeptide into a cellular host. Expression of the heterologous nucleicacid results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated chimeric pesticidal polypeptides may then be formulated inaccordance with conventional techniques for application to theenvironment hosting a target pest, e.g., soil, water, and foliage ofplants. See, for example EPA 0192319, and the references cited therein.

The novel chimeric pesticidal polypeptide-encoding polynucleotides andchimeric pesticidal polypeptides can be introduced into plant cells orplants to produce a transgenic plant that is resistant to pests that aresusceptible to the chimeric pesticidal polypeptide.

The novel chimeric pesticidal polypeptide-encoding polynucleotides canbe combined with constitutive, tissue-preferred or other promoters forexpression in plants. The promoters can be selected based on the desiredoutcome.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced chimericpesticidal polypeptide expression within a particular plant tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference.

Where low level expression is desired, weak promoters will be used.Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts. Alternatively, it is recognized that weakpromoters also encompasses promoters that are expressed in only a fewcells and not in others to give a total low level of expression. Where apromoter is expressed at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease expressionlevels.

Such weak constitutive promoters include, for example, the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also,U.S. Pat. No. 6,177,611, herein incorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Ad.USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

In one embodiment, the polynucleotide of interest is targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotide of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant or other organism. “Introducing” is intendedto mean presenting to the organism the polynucleotide or polypeptide insuch a manner that the sequence gains access to the interior of a cellof the organism. The methods of the invention do not depend on aparticular method for introducing a sequence into an organism, only thatthe polynucleotide or polypeptides gains access to the interior of atleast one cell of the organism. Methods for introducing polynucleotidesor polypeptides into plants are known in the art including, but notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. Nos.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the chimeric pesticidal polypeptide-encodingsequences of the invention can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, the introduction of the pesticidalprotein or variants and fragments thereof directly into the plant or theintroduction of the a pesticide-encoding transcript into the plant. Suchmethods include, for example, microinjection or particle bombardment.See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185;Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc.Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal ofCell Science 107:775-784, all of which are herein incorporated byreference. Alternatively, the pesticide-encoding polynucleotide can betransiently transformed into the plant using techniques known in theart. Such techniques include a viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use of particlescoated with polyethylimine.

In other embodiments, the novel chimeric pesticidal polypeptide-encodingpolynucleotide may be introduced into plants by contacting plants with avirus or viral nucleic acids. Generally, such methods involveincorporating a nucleotide construct within a viral DNA or RNA molecule.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

As used herein, the term plant also includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers, and the like. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introducedpolynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean andsugarcane plants are optimal, and in yet other embodiments corn plantsare optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

The present invention also provides a pesticidal composition comprisingan effective amount of at least one chimeric pesticidal polypeptide ofthe invention to reduce pest damage to the plant and methods ofprotecting a plant from a pest involving providing to the plant or tothe vicinity of the plant an effective amount of a pesticidalcomposition. By “effective amount” and “effective concentration” isintended an amount and concentration, respectively, that is sufficientto kill or inhibit the growth of a plant pest. Methods for determiningan “effective amount” and “effective concentration” are known to thoseof ordinary skill in art and include, for example, assays involvingplacing varying amounts of concentrations of the active ingredient(e.g., a chimeric pesticidal polypeptide) in contact with and/or in thevicinity of a pest and monitoring the survival and/or growth of the pestover time.

As described above, the effective amount of a chimeric pesticidalpolypeptide can vary depending on the formulation and method in whichthe formulation is applied to the plant or plant environment. As such,bacteria can be transformed with a nucleotide sequence encoding achimeric pesticidal polypeptide and can be used in the pesticidalcompositions as described herein. Thus, the pesticidal composition canbe an organism that is transformed to express a chimeric pesticidalpolypeptide. Alternatively, a chimeric pesticidal polypeptide can bepurified from the bacteria as described above. In some embodiments, thepesticidal composition or pesticidal formulation can include otheragricultural protectants, as described above. As used herein, the term“pesticidal composition” and “pesticidial formulation” are equivalentterms that have the same meaning unless indicated otherwise or apparentfrom the context as used.

As described above, the effective amount of a chimeric pesticidalpolypeptide can vary depending on the formulation and method in whichthe formulation is applied to the plant or plant environment. As such,bacteria can be transformed with a nucleotide sequence encoding achimeric pesticidal polypeptide and can be used in the pesticidalcompositions as described herein. Thus, the pesticidal composition canbe an organism that is transformed to express a chimeric pesticidalpolypeptide. Alternatively, the chimeric pesticidal polypeptide can bepurified from the bacteria as described above. In some embodiments, thepesticidal compositions or pesticidal formulation can include otheragricultural protectants, as described above.

As described above, the pesticidal composition can be, for example, adust, emulsion, solid (e.g., particle or pellets) or liquid.

The pesticidal composition can be provided to the plant by applying thepesticidal composition directly to the environment of the plant or tothe vicinity of the plant such as, for example, in the soil or othergrowth medium surrounding the plant to protect the plant from pestattacks. For example, the pesticidal composition can be applied directlyto the plant by atomizing, broadcasting, coating or pouring, dustingspraying, scattering, soil drenching, sprinkling or seed coating at thetime when the pest such as, for example, an insect pest has begun toappear on the plant or before the appearance of insect pests as aprotective measure.

Alternatively, the pesticidal composition can be introduced intoirrigation water and then applied to the plant during watering. It ispreferred to obtain good control of pest in the early stages of plantgrowth as this is the time when the plant can be most severely damaged.To maintain protection as plants grow and to obtain the greatestprotection of large plants, repeated applications of the pesticidalcomposition can be beneficial.

The number of applications and the rate of application depend on theintensity of infestation by the corresponding pest. When the pesticidalcomposition not only includes the chimeric pesticidal polypeptide and/ora chimeric pesticidal polypeptide-expressing bacteria, but also includesanother agricultural protectant, the formulation can be applied to thecrop area or plant to be treated simultaneously or in succession (i.e.,sequentially).

The pesticidal composition will reduce pest-related damage by at leastabout 5% to about 50%, at least about 10% to about 60%, at least about30% to about 70%, at least about 40% to about 80% or at least about 50%to about 90% or greater. Hence, the methods can be utilized to protectplants from pests. Protection may vary from a slight decrease in plantdamage caused by the pest (e.g., partial inhibition) to total decreasesuch that the plant is unaffected by the presence of the pest.

The present invention therefore provides methods of protecting plants,plant parts and plant host cells by providing a pesticidal compositioncomprising a chimeric pesticidal polypeptide.

In certain embodiments the polynucleotides of the present invention canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired trait. A trait, as used herein,refers to the phenotype derived from a particular sequence or groups ofsequences. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, such as other Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986)Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825,pentin (described in U.S. Pat. No. 5,981,722), and the like. Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the presentinvention can also be stacked with any other gene or combination ofgenes to produce plants with a variety of desired trait combinationsincluding, but not limited to, traits desirable for animal feed such ashigh oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids(e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802;and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); thedisclosures of which are herein incorporated by reference.

The polynucleotides of the present invention can also be stacked withtraits desirable for disease or herbicide resistance (e.g., fumonisindetoxification genes, U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE), and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs));the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides providing agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5.583,210), stalk strength, flowering time ortransformation technology traits such as cell cycle regulation or genetargeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); thedisclosures of which are herein incorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Various changes in phenotype are of interest including modifying thefatty acid composition in a plant, altering the amino acid content of aplant, altering a plant's pathogen defense mechanism, and the like.These results can be achieved by providing expression of heterologousproducts or increased expression of endogenous products in plants.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors in the plant. These changes result in a change in phenotypeof the transformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch, carbohydrateor nutrient metabolism as well as those affecting kernel size, sucroseloading, and the like.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated byreference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016,and the chymotrypsin inhibitor from barley, described in Williamson etal. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen et al. (1986)J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both ofwhich are herein incorporated by reference); and rice (Musumura et al.(1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Otheragronomically important genes encode latex, Floury 2, growth factors,seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene);glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360); or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments ±50%, in some embodiments±20%, in some embodiments ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods or employ the disclosed compositions.

Further, when an amount, concentration or other value or parameter isgiven as either a range, preferred range or a list of upper preferablevalues and lower preferable values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upperrange limit or preferred value and any lower range limit or preferredvalue, regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwise stated,the range is intended to include the endpoints thereof, and all integersand fractions within the range. It is not intended that the scope of thepresently disclosed subject matter be limited to the specific valuesrecited when defining a range.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Enhancement of the Insecticidal Activity of Cry ProteinsAgainst Western Corn Rootworms and Black Cutworms When Fused to MBP

A computer-designed and artificially synthesized Cry3Aa-type proteincalled IP3-1 (SEQ ID NO: 8), a truncated RX002 protein (SEQ ID NO: 12)cloned from a Bt strain, a shuffled Cry8Bb-type protein called 2A-12(SEQ ID NO: 10), and a truncated native Cry1Bd protein called 4c6 (SEQID NO: 14) were expressed in E. coli BL21 as fusion proteins comprisingan operably linked MBP. The amino acid sequences of the MBP-IP3-1,MBP-RX002, MBP-2A-12, and MBP-4c6 fusion proteins are set forth in SEQID NOS: 21, 23, 25, and 27, respectively. Nucleotide sequences encodingthe MBP-IP3-1, MBP-RX002, MBP-2A-12, and MBP-4c6 fusion proteins are setforth in SEQ ID NOS: 20, 22, 24, and 26, respectively.

To express MBP with these Bt Cry proteins, corresponding genes werecloned in an NEB pMAL vector and the proteins were purified usingamylose resin following the manufacturer's recommended method (NewEngland Biolabs, Inc., Ipswich, Mass., USA; Catalog No. E8021L). Thetarget protein eluted from amylase resin was concentrated in AmiconCentricon concentrator (10 kDa cutoff). The final fusion proteins weredissolved in 25 mM HEPES-NaOH buffer pH8 at a concentration around 2mg/ml. The protein concentration was determined by SDS-PAGE using bovineserum albumin as the reference. The pMAL vector has a protease sitespecific to Factor Xa that cleaves MBP and the linker off the Cryprotein. All Cry proteins included in this application were resistant toFactor Xa. The protease digestion was carried out in the HEPES buffer at1:25 protease and substrate ration at 20° C. for 16 hr. The digestionleading to the complete MBP removal was confirmed by SDS-PAGE.

MBP fusion and MBP free Cry proteins were diluted in 25 mM HEPES-NaOHbuffer pH 8 and 10 μL of diluted samples were mixed with 40 μL of moltenartificial insect diet made with low temperature melting agarose. Thediet mixture was then placed in each well of a 96-well micro-titer plateand allowed to feed with neonate insect larvae. After 4 days at 27° C.,the responses of insects towards the Cry proteins were scored using a0-3 numerical scoring system based on the size and mortality. If noresponse or normal growth was seen, Score 0 was given. When the growthwas somewhat retarded without any mortality, it was Score 1. Score 2meant partial death (multiple insects were used in each well) and stronggrowth inhibition. Score 3 indicated the complete mortality. Eachtreatment was repeated 6 times for possible highest score of 18 (3×6).In this scoring system, Score 9 with 6 repeats of one treatment meansthe 50% response (9 out of 18) of the treatment and called ILC50 (growthInhibition and Lethal Concentration for 50% response). The results ofthese assays are shown in Tables 3 and 4.

The results shown in Tables 3 and 4 with four different Cry proteinsdemonstrate that MBP-Cry fusion proteins have significantly increasedinsecticidal activity against both Coleopteran (Western Corn Rootworm)and Lepidopteran (Black Cutworm) insect species, when compared to theinsecticidial activity of their respective free Cry proteins. Theseresults further demonstrate the general applicability of the methods ofthe present invention to enhancing the insecticidal activity of Cryproteins. Table 3 shows ILC50 (based on the size or weight of the Cryprotein portion) values for insecticidal activity against Western CornRootworm of MBP-Cry Fusion Proteins and Free Cry Proteins.

TABLE 3 IP3-1 RX002 2A-12 MBP- free MBP- free MBP- free fusion Cryfusion Cry fusion Cry 7 ppm 103 ppm 89 ppm No activity* 32 ppm 152 ppm*Only an 11% response was observed at 2480 ppm.

Table 4 shows the ILC50 values for insecticidal activity against BlackCutworm of a MBP-4c6 Cry Fusion Protein and Free 4c6 Cry Protein.

TABLE 4 MBP-4c6 fusion free 4c6 75 ppm 148 ppm

EXAMPLE 2 MBP-Cry Fusion Protein With Increased Activity Against WCRW

A new vector, pMAL-SA (SEQ ID NO: 19), was constructed for makingMBP-Cry fusion proteins. This vector is based on NEB pMAL vector. It hasthe cry gene cloning site delineated with SphI and BamHI recognitionsequences at the end of MBP and a specially designed linker called “SA”linker between MBP and the Cry cloning site.

In order to clone a Bt cry gene, the cry coding region is amplified byPCR using appropriate forward and reverse primers. In the forwardprimer, there is an SphI site over the ATG translation initiation site.In the reverse primer, there is a stop codon at the end of the cry genecoding region and a BamHI site.

A computer-designed cry3Aa sequence, IP3-1, was synthesized and used asthe PCR template. The IP3-1 nucleotide sequence is set forth in SEQ IDNO: 7. This IP3-1 gene was cloned in pMAL-SA as described above toproduce a plasmid that contains MBP, the SA linker and IP3-1 codingregion (SEQ ID NO: 1). The second amino acid residue of IP3-1 wasmutated back to Asn from His to produce the MBP-SA-IP3-1 nucleotidesequence set forth in SEQ ID NO: 2.

The MBP-SA-IP3-1 fusion protein was expressed in E. coli BL21 andpurified by amylose resin affinity chromatography according to themethod described in Example 1. The column eluate was concentrated inAmicon Centricon and its buffer was exchanged to 25 mM HEPES-NaOH bufferpH 8. The fusion protein was digested with trypsin at 1:25trypsin-substrate ratio at 37° C. for 1 hr. Trypsin cleaves the proteinat the end of the linker to liberate the Cry protein from MBP and the SAlinker. IP3-1 was resistant to trypsin under this digestion condition.The digestion was confirmed with SDS-PAGE. Both the fusion and MBP-freeIP3-1 proteins were assayed against WCRW. Table 5 shows ILC50 values forinsecticidal activity against Western Corn Rootworm of a MBP-SA-IP3-1and MBP-free IP3-1. The assay results in Table 5 demonstrate that thereis a significant enhancement of WCRW activity when IP3-1 is operablelinked to MBP-SA (i.e., MBP-SA-IP3-1), when compared to the insecticidalactivity of MBP-free IP3-1 against WCRW.

TABLE 5 Protein ICL50 MBP-SA-IP3-1  6 ppm MBP-free IP3-1 269 ppm

EXAMPLE 3 Enhancement of Cry3Aa Protein Activity When Fused to NusA andTrx

A computer-designed and artificially synthesized cry3Aa-type gene calledIP3-1 was cloned in pET-43.1 EK/LIC for NusA fusion and pET-32 for TrxAfusion by following the manufacturer's directions (EMD Biosciences,Madison, Wis., USA). These vectors were used to express NusA-IP3-1 andTrxA-IP3-1 fusion proteins. The amino acid sequences of the NusA-IP3-1and TrxA-IP3-1 fusion proteins are set forth in SEQ ID NOS: 16 and 18,respectively. Nucleotide sequences encoding the NusA-IP3-1 andTrxA-IP3-1 fusion proteins are set forth in SEQ ID NOS: 15 and 17,respectively. The amino acid sequence of IP3-1 is set forth in SEQ IDNO: 8. A nucleotide sequence encoding IP3-1 is set forth in SEQ ID NO:7.

The fusion proteins were purified by affinity chromatography usingNi-NTA agarose (Qiagen Inc., Valencia, Calif., USA) according to themanufacturer's directions. The Cry3Aa protein was then digested awayfrom the tags including NusA, TrxA, 6XHis etc. with enterokinase and theinsecticidal activity of the free Cry3A protein was compared with itsNusA and TrxA fusion proteins. The insect assay was conducted using WCRWas described above. Table 6 shows the ILC50 values for insecticidalactivity against Western Corn Rootworm of a MBP-SA-IP3-1 and MBP-freeIP3-1 protein. The IP3-1 protein sample was produced from theNusA-Cry3Aa fusion by enterokinase digestion. As demonstrated in theresults in Table 6, both the NusA-IP3-1-and TrxA-IP3-1-fusion proteins,which comprise a solubility-enhancing polypeptide and a Cry3A protein(IP3-1), have an increased insecticidal activity against WCRW asevidenced by the significantly lower ICL50 values, when compared toinsecticidal activity of free IP3-1.

TABLE 6 Protein ICL50 NusA-IP3-1-fusion  26 ppm TrxA-IP3-1-fusion  31ppm Tag free IP3-1 554 ppm

EXAMPLE 4 Generation of a MBP-SA-RX002 Transformation Construct

The maltose-binding protein (MBP-SA) (SEQ ID NO: 4) was fused to theN-terminus of RX002 (SEQ ID NO:11) in a synthetic gene designed for thisinvention (SEQ ID NO:32 encoding SEQ ID NO:33). The gene was cloned as aBamHI—Stul fragment into a Gateway entry vector containing a plantexpression cassette with the BSV(AY) TR PROMOTER—ADH1 INTRON1 sequenceand the potato PIN II terminator sequence. The resulting plantexpression cassette contains the following components operatively linkedtogether in this order; BSV (AY) TR PRO-ADH1-INTRON1, the MBP-SA-RX002gene, and the PIN II terminator. The expression cassette is flanked byGateway attL3 and attL4 recombination sites and this entry vector wasused to transfer the expression cassette into an attR3 and attR4containing binary destination transformation vector. The finaltransformation vector contains the MBP-SA-RX002 expression cassetteupstream of a cassette containing the maize Ubiquitin1promoter-5′UTR-Ubiquitin intron1 controlling expression of a PATselectable marker gene with the 35S terminator sequence.

EXAMPLE 5 Agrobacterium-Mediated Transformation of Maizeand Regenerationof Transgenic Plants

For Agrobacterium-mediated transformation of maize with a promotersequence of the invention, the method of Zhao was employed (U.S. Pat.No. 5,981,840, and PCT patent publication WO98/32326; the contents ofwhich are hereby incorporated by reference). Briefly, immature embryoswere isolated from maize and the embryos contacted with a suspension ofAgrobacterium under conditions whereby the bacteria were capable oftransferring the promoter sequence of the invention to at least one cellof at least one of the immature embryos (step 1: the infection step). Inthis step the immature embryos were immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos wereco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos were cultured on solid mediumfollowing the infection step. Following the co-cultivation period and anoptional “resting” step was performed. In this resting step, the embryoswere incubated in the presence of at least one antibiotic known toinhibit the growth of Agrobacterium without the addition of a selectiveagent for plant transformants (step 3: resting step). The immatureembryos were cultured on solid medium with antibiotic, but without aselecting agent, for elimination of Agrobacterium and for a restingphase for the infected cells. Next, inoculated embryos were cultured onmedium containing a selective agent and growing transformed callus wasrecovered (step 4: the selection step). The immature embryos werecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus was then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium were cultured on solid medium to regenerate the plants.

EXAMPLE 6 Expression of MBP-RX002 Fusion in Transgenic Maize Tissue

Transgenic events derived from the testing vector were evaluated forexpression of MBP-RX002 by Western analysis. Leaf and root material fortransgenic maize expressing MBP-RX002 were lyophilized then powderedwith 5/32 inch BBs (i.e., birdshot) using a Geno/Grinder 2000homogenizer at 1700 beats per minute for 30 seconds. 80 μL of grindingbuffer (1×PBS+0.1% Tween-20+1% 2-mercaptoethanol containing RochecOmplete protease inhibitor (Roche Applied Science, Indianapolis, Ind.,USA; Catalog No. 04693124001; at one tablet per 7 mL) was added to eachsample. Pulverization was repeated for an additional 30 seconds, thenthe samples were sonicated for 5 minutes at room temperature in a VWR75D sonicator. After centrifugation at 21,000 g/4° C. for 15 minutes,supernatants were collected. Protein concentrations were determined fromthe supernatants using Thermo Scientific Coomassie Plus Kit (23236) anda SpectraMAX 190 spectrophotometer. Samples were normalized for totalprotein in 21 μL using grinding buffer as diluent. Seven microliters of4× LDS dye containing 1% 2-mercaptoethanol was added to each sampleprior to heating at 80° C. for 10 minutes. Twenty-five microliters ofsample was loaded per lane on a NuPAGE Novex 4-12% Bis-Tris midi gel(Invitrogen WB1402BOX) and electrophoresed at 200 V for 1 hour. Proteinwas transferred to a nitrocellulose membrane using an Invitrogen iBlotwith a transfer stack (IB301001). The membrane was blocked for 30minutes in 1×PBS+0.1% Tween-20+5% powdered milk w/v (blocking buffer),then incubated overnight at 4° C. with rabbit polyclonal antibodyagainst RX002, diluted in blocking buffer at 1:4000. Membrane was washed4×5 minutes in PBST (1×PBS+0.1% Tween-20), then incubated for 2 hourswith goat anti-rabbit HRP conjugated secondary antibody (Pierce 31460;10 μg/mL working stock) at a 1:5000 dilution in blocking buffer. Themembrane was washed 4×5 minutes in PBST and developed using Thermo SuperSignal West Dura Extended Duration Substrate (34076). Visualization ofhybridization signal was accomplished using a Fujifilm LAS-4000 imagingsystem.

The results of this analysis are shown in Table 7. Accumulation ofMBP-RX002 was detected in 8 of the 10 events sampled for Westernanalysis in either root or leaf and root tissue. An 118 kD protein bandcorresponding to the expected size of the MBP-RX002 fusion was observedin both leaf and root or root tissue demonstrating that the fusion canbe expressed in planta. In addition to the full length protein, a 75 kDimmunoreactive protein band corresponding to the expected size of RX002was also observed in some events indicating that some proportion of thefull-length fusion protein was processed in planta to release RX002.

TABLE 7 Event No. Expression in leaf Expression in root Control − −123434603 − − 123434607 +++ ++++ 123434610 − − 123434613 ++++ ++++123434614 − + 123434615 − ++ 123434616 ++++ ++++ 123434617 ++++ ++++123434618 ++++ ++++ 123434619 − +++

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method of enhancing pesticidal activity of aCry endotoxin, the method comprising operably linking a first amino acidsequence of a solubility-enhancing polypeptide to a second amino acidsequence of a Cry endotoxin, whereby a chimeric pesticidal polypeptideis produced, the chimeric pesticidal polypeptide comprising the firstamino acid sequence operably linked to second amino acid sequence. 2.The method of claim 1; wherein the solubility-enhancing polypeptide isselected from the group consisting of maltose-binding protein (MBP),thioredoxin, and transcription elongation factor NusA.
 3. The method ofclaim 2, wherein the solubility-enhancing polypeptide comprises an aminoacid sequence selected from the group consisting of the amino acidsequences set forth in SEQ ID NOS: 4, 6, 34, 35 and
 36. 4. The method ofclaim 1, 2 or 3, wherein the Cry endotoxin comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 8, 10, 12 and14.
 5. The method of claim 1, 2, 3 or 4, wherein the chimeric pesticidalpolypeptide comprises an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOS: 2, 16,18, 21, 23, 25, 27 and
 33. 6. A chimeric pesticidal polypeptidecomprising a first amino acid sequence of a solubility-enhancingpolypeptide operably linked to a second amino acid sequence of a Cryendotoxin, wherein the operably linked first and second amino sequencescomprise an amino acid sequence selected from the group consisting of:(a) the amino acid sequence set forth in SEQ ID NO: 2, 16, 18, 21, 23,25, 27 or 33; and (b) an amino acid sequence encoded by the nucleotidesequence set forth in SEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or
 32. 7. Anucleic acid molecule encoding the chimeric pesticidal polypeptide ofclaim
 6. 8. An expression cassette comprising a promoter that drivesexpression in a host cell operably linked to the nucleic acid moleculeof claim
 7. 9. A vector comprising the expression cassette of claim 8.10. A transformed plant, plant part, plant cell or seed comprising inits genome the expression cassette of claim
 8. 11. A pesticidalcomposition comprising an effective amount of a chimeric pesticidalpolypeptide of claim 6 or an active variant or fragment thereof havingpesticidal activity.
 12. A method of protecting a plant from an insectpest, the method comprising providing an effective amount of thepesticidal composition of claim 10 to reduce insect pest-related damageto the plant.
 13. A plant comprising a polynucleotide construct stablyincorporated in its genome, the polynucleotide construct comprising anucleotide sequence operably linked to a promoter that drives expressionin the plant, wherein the nucleotide sequence encodes a chimericpesticidal polypeptide comprising a first amino acid sequence of asolubility-enhancing polypeptide operably linked to a second amino acidsequence of a Cry endotoxin, wherein the solubility-enhancingpolypeptide is selected from the group consisting of maltose-bindingprotein (MBP), thioredoxin, and transcription elongation factor NusA.14. The plant of claim 13, wherein the nucleotide sequence is selectedfrom the group consisting of: (a) the nucleotide sequence set forth inSEQ ID NO: 1, 15, 17, 20, 22, 24, 26 or 32; and (b) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 16,18, 21, 23, 25, 27 or
 33. 15. A method of protecting a plant, plant partor plant host cell from an insect pest, the method comprising the stepsof: (a) introducing into the plant, plant part or plant host cell anexpression cassette of claim 7; and (b) regenerating the plant, plantpart or plant host cell into a morphologically normal fertile plant,wherein the plant or part thereof comprises a chimeric pesticidalpolypeptide.
 16. A method of enhancing the resistance of a plant to atleast one pest, the method comprising introducing into a plant or atleast one plant cell a polynucleotide construct comprising a nucleotidesequence operably linked to a promoter that drives expression in theplant, wherein the nucleotide sequence encodes a chimeric pesticidalpolypeptide comprising a first amino acid sequence of asolubility-enhancing polypeptide operably linked to a second amino acidsequence of a Cry endotoxin.
 17. The method of claim 16, wherein thesolubility-enhancing polypeptide is selected from the group consistingof maltose-binding protein (MBP), thioredoxin, and transcriptionelongation factor NusA.
 18. The method of claim 17, wherein thenucleotide sequence comprises a nucleotide sequence selected from thegroup consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:1, 15, 17, 20, 22, 24, 26 or 32; and (b) a nucleotide sequence encodingan amino acid sequence comprising the amino acid sequence set forth inSEQ ID NO: 2, 16, 18, 21, 23, 25, 27 or 33.